Muteins of Tear Lipocalin

ABSTRACT

The present invention relates to novel muteins derived from tear lipocalin or a homologue thereof. In particular, the invention relates to a mutein of human tear lipocalin. The invention also refers to a corresponding nucleic acid molecule encoding such a mutein and to a method for its generation. The invention further refers to a method for producing such a mutein. Finally, the invention is directed to a pharmaceutical composition comprising such a lipocalin mutein as well as to various use of the mutein.

The present invention relates to novel muteins derived from tearlipocalin or a homologue thereof. In particular, the invention relatesto a mutein of human tear lipocalin. The invention also refers to acorresponding nucleic acid molecule encoding such a mutein and to amethod for its generation. The invention further refers to a method forproducing such a mutein. Finally, the invention is directed to apharmaceutical composition comprising such a lipocalin mutein as well asto various use of the mutein.

The members of the lipocalin protein family (Pervaiz, S., and Brew, K.(1987) FASEB J. 1, 209-214) are typically small, secreted proteins whichare characterized by a range of different molecular-recognitionproperties: their ability to bind various, principally hydrophobicmolecules (such as retinoids, fatty acids, cholesterols, prostaglandins,biliverdins, pheromones, tastants, and odorants), their binding tospecific cell-surface receptors and their formation of macromolecularcomplexes. Although they have, in the past, been classified primarily astransport proteins, it is now clear that the lipocalins fulfill avariety of physiological functions. These include roles in retinoltransport, olfaction, pheromone signaling, and the synthesis ofprostaglandins. The lipocalins have also been implicated in theregulation of the immune response and the mediation of cell homoeostasis(reviewed, for example, in Flower, D. R. (1996) Biochem. J. 318, 1-14and Flower, D. R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).

The lipocalins share unusually low levels of overall sequenceconservation, often with sequence identities of less than 20%. In strongcontrast, their overall folding pattern is highly conserved. The centralpart of the lipocalin structure consists of a single eight-strandedanti-parallel β-sheet closed back on itself to form a continuouslyhydrogen-bonded β-barrel. One end of the barrel is sterically blocked bythe N-terminal peptide segment that runs across its bottom as well asthree peptide loops connecting the β-strands. The other end of theβ-barrel is open to the solvent and encompasses a target-binding site,which is formed by four peptide loops. It is this diversity of the loopsin the otherwise rigid lipocalin scaffold that gives rise to a varietyof different binding modes each capable of accommodating targets ofdifferent size, shape, and chemical character (reviewed, e.g., inFlower, D. R. (1996), supra; Flower, D. R. et al. (2000), supra, orSkerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).

Human tear pre-albumin, now called tear lipocalin (TLPC), was originallydescribed as a major protein of human tear fluid (approximately onethird of the total protein content) but has recently also beenidentified in several other secretory tissues including prostate, nasalmucosa and tracheal mucosa. Homologous proteins have been found in rat,pig, dog and horse. Tear lipocalin is an unusual lipocalin memberbecause of its high promiscuity for relative insoluble lipids andbinding characteristics that differ from other members of this proteinfamily (reviewed in Redl, B. (2000) Biochim. Biophys. Acta 1482,241-248). A remarkable number of lipophilic compounds of differentchemical classes such as fatty acids, fatty alcohols, phospholipids,glycolipids and cholesterol are endogenous ligands of this protein.Interestingly, in contrast to other lipocalins the strength of ligand(target) binding correlates with the length of the hydrocarbon tail bothfor alkyl amides and fatty acids. Thus, tear lipocalin binds moststrongly the least soluble lipids (Glasgow, B. J. et al. (1995) Curr.Eye Res. 14, 363-372; Gasymov, O. K. et al. (1999) Biochim. Biophys.Acta 1433, 307-320).

The precise biological function of human tear lipocalin has not beenfully elucidated so far and is still a matter of controversy. In tearfluid, it appears to be most important for the integrity of the tearfilm by removing lipids from the mucous surface of the eye to the liquidphase (reviewed in Gasymov, O. K. et al. (1999), supra). However, itdisplays additional activities in vitro that are very unusual amonglipocalins, namely inhibition of cystein proteinases as well asnon-specific endonuclease activity (van't Hof, W. et al. (1997) J. Biol.Chem. 272, 1837-1841; Yusifov, T. N. et al. (2000) Biochem. J. 347,815-819). Recently, it has been demonstrated that tear lipocalin is ableto bind several lipid peroxidation products in vitro resulting in thehypothesis that it might function as a physiologicaloxidative-stress-induced scavenger of potentially harmful lipophilicmolecules (Lechner, M. et al. (2001) Biochem. J. 356, 129-135).

Proteins, which selectively bind to their corresponding targets by wayof non-covalent interaction, play a crucial role as reagents inbiotechnology, medicine, bioanalytics as well as in the biological andlife sciences in general. Antibodies, i.e. immunoglobulins, are aprominent example of this class of proteins. Despite the manifold needsfor such proteins in conjunction with recognition, binding and/orseparation of ligands/targets, almost exclusively immunoglobulins arecurrently used. The application of other proteins with definedligand-binding characteristics, for example the lectins, has remainedrestricted to special cases.

Rather recently, members of the lipocalin family have become subject ofresearch concerning proteins having defined ligand-binding properties.The PCT publication WO 99/16873 discloses the class of so-calledAnticalins®; i.e. polypeptides of the lipocalin family with mutatedamino acid positions in the region of the four peptide loops, which arearranged at the end of the cylindrical β-barrel structure encompassingthe binding pocket, and which correspond to those segments in the linearpolypeptide sequence comprising the amino acid positions 28 to 45, 58 to69, 86 to 99, and 114 to 129 of the bilin-binding protein of Pierisbrassicae. The PCT publication WO 00/75308 discloses muteins of thebilin-binding protein, which specifically bind digoxigenin, whereas theInternational Patent Applications WO 03/029463 and WO 03/029471 relateto muteins of the human neutrophil gelatinase-associated lipocalin andapolipoprotein D, respectively. In order to further improve and finetune ligand affinity, specificity as well as folding stability of alipocalin variant various approaches using different members of thelipocalin family have been proposed (Skerra, A. (2001) Rev. Mol.Biotechnol. 74, 257-275; Schlehuber, S., and Skerra, A. (2002) Biophys.Chem. 96, 213-228), such as the replacement of additional amino acidresidues.

However, for various applications it could also be advantageous to havemore than one binding site per molecule available—either the naturalbinding pocket plus an engineered additional (protein)-binding site ortwo different engineered binding sites. For example, it could beconsidered to use lipocalin muteins as adapter or linker molecules whichmay be attached to a given binding partner via binding site I, whereasbinding site II is used for screening/selection purposes or the like.One possibility to achieve this goal is the use of fusion proteinscomprising two lipocalin muteins of same or different bindingspecificity, which are coupled to each other by a peptide linker. Suchfusion proteins, also called “duocalins”, are described in WO 99/16873and also by Schlehuber, S., and Skerra, A. (2001), Biol. Chem. 382,1335-1342, for example.

Recently high-affinity histamine-binding proteins have been identifiedin the saliva of Rhipicephalus appendiculatus ticks (Paesen, G. C. etal. (1999) Mol. Cell 3, 661-671). These proteins sequester histamine atthe wound site, outcompeting histamine receptors for the ligand in orderto suppress inflammation during blood feeding. The crystal structure ofthese histamine-binding proteins has revealed a lipocalin fold novel incontaining two binding sites for histamine having different bindingaffinities. The sites, one of which is a typical lipocalin binding site,are orthogonally arranged and highly rigid, forming an unusually polarinternal surface that specifically complements the molecular propertiesof histamine. A related protein termed SHBP, which is secreted by arodent- and cattle-feeding tick, binds both histamine and serotonin atthe two different binding sites (Sangamnatdej, S. et al. (2002) InsectMol. Biol. 11, 79-86). The high-affinity binding site lies perpendicularto the long axis of the β-barrel leading to distortions in the proteinstructure compared with other lipocalins. Thus, it appears as if such abinding site cannot be engineered in any given lipocalin. On the otherhand, since the binding sites are rather buried in the core of theβ-barrel there appear to be sterical limitations with regard to ligandsize.

Thus, there remains a need for the generation of binding proteins thatuses different binding sites and/or alternative lipocalin scaffolds,simply for the reason to have more options for practical realisation.

Accordingly, it is an object of the invention to provide alternativelipocalin muteins having binding affinity to a given target.

This object is accomplished by a lipocalin mutein having the features ofthe independent claims as well as the method for its generation.

In one embodiment such a lipocalin mutein is a mutein derived from apolypeptide of tear lipocalin or a homologue thereof, wherein the muteincomprises at least two mutated amino acid residues at any sequenceposition in the N-terminal peptide stretch and the three peptide loopsBC, DE, and FG (cf. FIG. 2) arranged at the end of the β-barrelstructure that is located opposite to the natural lipocalin bindingpocket, wherein said tear lipocalin or homologue thereof has at least60% sequence homology with human tear lipocalin, and wherein the muteinbinds a given target with detectable affinity.

In more illustrative terms, this embodiment is based on the finding ofthe inventors that amino acids in the three loops at the closed end ofthe internal ligand binding site of a tear lipocalin and/or theN-terminal peptide stretch of the tear lipocalin (cf. FIG. 1) can bemutated in order to obtain lipocalin muteins that bind a given targetwith determinable affinity. Thus, the invention provides a structurallynew class of lipocalin muteins with antibody-like binding properties.This means that these muteins can be used in the same way for thegeneration of new binding proteins with a predetermined specificity asthe class of the above mentioned so-called Anticalins® (lipocalinmuteins which are derived from the proteins of the lipocalin family suchas the bilin-binding protein of Pieris brassicae, in which amino acidpositions in the four peptide loops positioned at the open end of theligand binding site are mutated). For this reason, these new lipocalinmuteins of the present invention are also considered to belong to theselipocalin muteins designated Anticalins®.

In another embodiment, a mutein of the invention is also a muteinderived from a polypeptide of tear lipocalin or a homologue thereof,wherein the mutein comprises at least two mutated amino acid residues atany sequence position in the four peptide loops AB, CD, EF, and GH (cf.FIG. 2) encompassing the natural lipocalin binding pocket, wherein saidtear lipocalin or homologue thereof has at least 60% sequence homologywith human tear lipocalin, and wherein the mutein binds a given targetwith detectable affinity. Accordingly, this embodiment provides for anew class of scaffold in which amino acids in the four loops at the openend of the ligand binding site of the lipocalins can be mutated for thegeneration of binding molecules against a desired target.

In yet another embodiment the invention relates to a mutein derived froma polypeptide of tear lipocalin or a homologue thereof,

-   -   wherein the mutein comprises at least two mutated amino acid        residues at any sequence position in the N-terminal region and        the three peptide loops BC, DE, and FG arranged at the end of        the β-barrel structure that is located opposite to the natural        lipocalin binding pocket,    -   wherein the mutein comprises at least two mutated amino acid        residues at any sequence position in the four peptide loops AB,        CD, EF, and GH encompassing the natural lipocalin binding        pocket,    -   wherein said tear lipocalin or homologue thereof has at least        60% sequence homology with human tear lipocalin, and    -   wherein the mutein binds at least one given target with        detectable affinity.

Thus, the invention also provides for the first time a monomericlipocalin mutein (Anticalin®) that due to the presence of two bindingsites can have binding specifity for two given ligands. Such abispecific molecule can be considered to be functionally equivalent to abispecific antibody molecule such as a bispecific diabody. However,compared to a bispecific diabody (or antibody fragment in general), thisnew class of bispecific lipocalin muteins (Anticalins®) has theadvantage that it is composed only of one polypeptide chain whereas adiabody consists of two polypeptide chains that are non-covalentlyassociated with each other.

A bispecific lipocalin mutein of this new class of binding proteins maybe used as an adapter molecule. For example, when having bindingaffinity to two different receptors, such a bispecific lipocalinmolecule can cross-link these receptors. An example of such a lipocalinmutein (Anticalin®) would be a mutein, wherein the first binding sitebinds to an apoptose receptor such as the CD95 (also known as Fas or Apo1 receptor) and the second binding site can bind to a cell surfacereceptor, which is expressed on the same cell. Binding of such abispecific mutein in a bicellular manner may result in mutualcross-linking of the CD95 apoptose receptor and the second cell surfacereceptor target antigen, which can effectively induce apoptosis of thecells (cf. Jung, G. et al. (2001) Cancer Res. 61, 1846-1848). Howeversuch a bispecific mutein may also have only binding affinity for onegiven target. Such a mutein may be useful as a molecular storage fordrugs that are to be slowly released into the blood stream.

The term “mutagenesis” as used herein means that the experimentalconditions are chosen such that the amino acid naturally occurring at agiven sequence position of the lipocalin used can be substituted by atleast one amino acid that is not present at this specific position inthe respective natural polypeptide sequence. The term “mutagenesis” alsoincludes the (additional) modification of the length of sequencesegments by deletion or insertion of one or more amino acids. Thus, itis within the scope of the invention that, for example, one amino acidat a chosen sequence position is replaced by a stretch of three randommutations, leading to an insertion of two amino acid residues comparedto the length of (the respective segment) of the wild type protein. Suchan insertion of deletion may be introduced independently from each otherin any of the peptide segments that can be subjected to mutagenesis inthe invention. In one exemplary embodiment of the invention, aninsertion of several mutations is introduced in the loop AB of theselected lipocalin scaffold (cf. Examples 2 and 28, respectively). Theterm “random mutagenesis” means that no predetermined single amino acid(mutation) is present at a certain sequence position but that at leasttwo amino acids can be incorporated into a selected sequence positionduring mutagenesis with a certain probability.

Such experimental conditions can, for example, be achieved byincorporating codons with a degenerate base composition into anucleotide acid encoding the respective lipocalin employed. For example,use of the codon NNK or NNS (wherein N=adenine, guanine or cytosine orthymine; K=guanine or thymine; S=adenine or cytosine) allowsincorporation of all 20 amino acids plus the amber stop codon duringmutagenesis, whereas the codon VVS limits the number of possiblyincorporated amino acids to 12, since it excludes the amino acids Cys,Ile, Leu, Met, Phe, Trp, Tyr, Val from being incorporated into theselected position of the polypeptide sequence; use of the codon NMS(wherein M=adenine or cytosine), for example, restricts the number ofpossible amino acids to 11 at a selected sequence position since itexcludes the amino acids Arg, Cys, Gly, Ile, Leu, Met, Phe, Trp, Valfrom being incorporated at a selected sequence position. In this respectit is noted that codons for other amino acids (than the regular 20naturally occurring amino acids) such as selenocystein or pyrrolysinecan also be incorporated into a nucleic acid of a mutein. It is alsopossible, as described by Wang, L., et al. (2001) Science 292, 498-500,or Wang, L., and Schultz, P. G. (2002) Chem. Commun. 1, 1-11, to use“artificial” codons such as UAG which are usually recognized as stopcodons in order to insert other unusual amino acids, for exampleo-methyl-L-tyrosine or p-aminophenylalanine.

The term “tear lipocalin” as used herein is not limited to the humantear lipocalin (SWISS-PROT Data Bank Accession Number M90424) but isintended to include all polypeptides having the structurally conversedlipocalin fold as well as a sequence homology or a sequence identitywith respect to the amino acid sequence of the human tear lipocalin ofat least 60%. The term lipocalin fold is used in its regular meaning asused, e.g., in Flower, D. R. (1996), supra, to describe the typicalthree-dimensional lipocalin structure with a conformationally conservedβ-barrel as a central motif made of a cylindrically closed β-sheet ofeight antiparallel strands, wherein the open end of the barrel theβ-strands are connected by four loops in a pairwise manner so that thebinding pocket is formed (see also FIG. 2).

The definition of the peptide loops as used in the present invention isalso in accordance with the regular meaning of the term lipocalin foldand is as follows and also illustrated in FIG. 2: The peptide loop(segment) AB connects the β-strands A and B of the cylindrically closedβ-sheet, the peptide loop CD connects the β-strands C and D, the peptideloop EF connects the β-strands E and F, the peptide loop GH connects theβ-strands G and H, the peptide loop BC connects the β-strands B and C,the loop DE connects the β-strands D and E, and the loop FG connects theβ-strands F and G. As can be seen from FIG. 2 the loops AB, CD, EF andGH form the known binding site of the lipocalins (which was thereforecalled the open end), whereas, as found in the present invention, theloops BC, DE and FG can be used together with the N-terminal peptidestretch to form a second binding site which is located at the closed endof the β-barrel.

In accordance with the above, the term “tear lipocalin” includesstructural homologues, already identified or yet to be isolated, fromother species which have an amino acid sequence homology or sequenceidentity of more than about 60%. The term “homology” as used herein inits usual meaning and includes identical amino acids as well as aminoacids which are regarded to be conservative substitutions (for example,exchange of a glutamate residue by a aspartate residue) at equivalentpositions in the linear amino acid sequence of two proteins that arecompared with each other. The term “sequence identity” or “identity” asused in the present invention means the percentage of pair-wiseidentical residues—following homology alignment of a sequence of apolypeptide of the present invention with a sequence in question—withrespect to the number of residues in the longer of these two sequences.

The percentage of sequence homology or sequence identity is determinedherein using the program BLASTP, version blastp 2.2.5 (Nov. 16, 2002;cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402). Thepercentage of homology is based on the alignment of the entirepolypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1; cutoff valueset to 10⁻³) including the propeptide sequences, using the human tearlipocalin as reference in a pairwise comparison. It is calculated as thepercentage of numbers of “positives” (homologous amino acids) indicatedas result in the BLASTP program output divided by the total number ofamino acids selected by the program for the alignment. It is noted inthis connection that this total number of selected amino acids candiffer from the length of the tear lipocalin (176 amino acids includingthe propeptide) as it is seen in the following.

Examples of homologues proteins are Von Ebners gland protein 1 of Rattusnorvegicus (VEGP protein; SWISS-PROT Data Bank Accession Numbers P20289)with a sequence homology of ca. 70% (125 positives/178 positionsincluding the propeptide; when the 18 residues long propeptidescontaining 13 “positives” are not taken into account: 112 positives/160,resulting also in an homology of ca. 70%), Von Ebners gland protein 2 ofRattus norvegicus (VEG protein 2; SWISS-PROT Data Bank Accession NumbersP41244) with a sequence homology of ca. 71% (127 positives/178 includingthe propeptide; when the 18 residues long propeptides are not taken intoaccount: 114 positives/160, the homology is determined to be also ca.71%), Von Ebners gland protein 2 of Sus scrofra (pig) (LCN1; SWISS-PROTData Bank Accession Numbers P53715) with a sequence homology of about74% (131 positives/176 positions including the propeptide; when the 18residues long propeptides containing 16 “positives” are not taken intoaccount: 115 positives/158, resulting in an homology of ca. 73%), or theMajor allergen Can f1 precursor of dog (ALL 1, SWISS-PROT Data BankAccession Numbers 018873) with a sequence homology of ca. 70%, (122positives/174 positions, or 110 positives/156=ca. 70% homology, when thepropeptides with 12 positives are excluded) as determined with theprogram BLASTP as explained above. Such a structural homologue of thetear lipocalin can be derived from any species, i.e. from prokaryotic aswell as from eukaryotic organisms. In case of eukaryotic organisms, thestructural homologue can be derived from invertebrates as well asvertebrates such as mammals (e.g., human, monkey, dog, rat or mouse) orbirds or reptiles.

In case a protein other than tear lipocalin is used in the presentinvention, the definition of the mutated sequence positions given fortear lipocalin can be assigned to the other lipocalin with the help ofpublished sequence alignments or alignments methods which are availableto the skilled artisan. A sequence alignment can, for example, becarried out as explained in WO 99/16873 (cf. FIG. 3 therein), using anpublished alignment such as the one in FIG. 1 of Redl, B. (2000)Biochim. Biophys. Acta 1482, 241-248. If the three-dimensional structureof the lipocalins are available structural superpositions can also beused for the determination of those sequence positions that are to besubjected to mutagenesis in the present invention. Other methods ofstructural analysis such as multidimensional nuclear magnetic resonancespectroscopy can also be employed for this purpose.

The homologue of tear lipocalin can also be a mutein protein of tearlipocalin itself, in which amino acid substitutions are introduced atpositions other than the positions selected in the present invention.For example, such a mutein can be a protein in which positions at thesolvent exposed surface of the β-barrel are mutated compared to the wildtype sequence of the tear lipocalin in order to increase the solubilityor the stability of the protein.

In general, the term “tear lipocalin” includes all proteins that have asequence homology or sequence identity of more than 60%, 70% 80%, 85%,90%, or 95% in relation to the human tear lipocalin (SWISS-PROT DataBank Accession Number M90424).

In one preferred embodiment of the invention the mutein as disclosedherein is derived from human tear lipocalin. In other preferredembodiments the mutein is derived from the VEGP protein, VEG protein 2,LCN1, or ALL 1 protein.

If the binding site at the closed end of the β-barrel is used, themutein according to the invention typically comprises mutations at anytwo or more of the sequence positions in the peptide segmentscorresponding to the sequence positions 7-14, 41-49, 69-77, and 87-98 ofthe linear polypeptide sequence of human tear lipocalin. The positions7-14 are part of the N-terminal peptide stretch, the positions 41-49 arecomprised in the BC loop, the positions 60-77 are comprised in the DEloop and the positions 87-98 are comprised in the FG loop.

In more specific embodiments of those muteins the mutations areintroduced at those sequence positions, which correspond to thepositions 8, 9, 10, 11, 12, 13, 43, 45, 47, 70, 72, 74, 75, 90, 92, 94,and 97 of human tear lipocalin. Usually, such a mutein comprisesmutations at 5-10 or 12-16 or all 17 of the sequence positions.

In case the binding site at the open end of the β-barrel is subjected tomutagenesis a lipocalin mutein according to the invention comprisesmutations at any two or more of the sequence positions in the peptidesegments corresponding to the sequence positions 24-36, 53-66, 79-84,and 103-110 of the linear polypeptide sequence of human tear lipocalin.The positions 24-36 are comprised in the AB loop, the positions 53-66are comprised in the CD loop, the positions 69-77 are comprised in theEF loop and the positions 103-110 are comprised in the GH loop. In oneembodiment of the invention, an insertion of 1 to 6 amino acid residues,preferably of 2 to 4 amino acid residues, is introduced into the peptidesegment hat is formed by the sequence positions corresponding tosequence positions 24-36 of human tear lipocalin. This insertion can beincluded at any position within this segment. In one exemplaryembodiment, this insertion is introduced between sequence positions 24and 25 of human tear lipocalin. However, it is also noted again that theintroduction of a stretch of at least two amino acids into a peptidesegment that is part of the binding sites used here, is not limited tothe segment comprising residues 24-26 but can be included in any segmentparticipating in the formation of one of the two binding sites chosenherein.

Accordingly, a mutein having two binding sites comprises mutations atany two or more of the sequence positions in the peptide segmentscorresponding to the sequence positions 7-14, 41-49, 69-77, and 87-97 ofthe linear polypeptide sequence of human tear lipocalin and additionalmutations at any two or more of the sequence positions in the peptidesegments corresponding to the sequence positions 24-36, 53-66, 79-84,and 103-110 of the linear polypeptide sequence of human tear lipocalin.

In this respect it is noted that the number of the segments (loops)defined above which are used for mutagenesis can vary (the N-terminalpeptide stretch is included in the meaning of the term segment or loop).It is not necessary to mutate all four of these segments all together ofeach of the two binding sites, for example in a concerted mutagenesis.But it is also possible to introduce mutations only in one, two or threesegments of each binding site in order to generate a mutein havingdetectable affinity to a given target. Therefore, it is possible tosubject, for example, only two or three segments at the closed end ofthe β-barrel to mutagenesis if a binding molecule with only oneengineered binding site is wanted. If this molecule is then wanted tohave binding affinity towards a second target, sequence positions in anyof the four loops of the second binding site can then be mutated. It isalso possible, however, to mutate peptide loops of both binding sites,even if a given target is to be bound by one of the binding site only.

The lipocalin muteins of the invention may comprise the wild type(natural) amino acid sequence outside the mutated segments. On the otherhand, the lipocalin muteins disclosed herein may also contain amino acidmutations outside the sequence positions subjected to mutagenesis aslong as those mutations do not interfere with the binding activity andthe folding of the mutein. Such mutations can be accomplished veryeasily on DNA level using established standard methods (Sambrook, J. etal. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.). Possible alterationsof the amino acid sequence are insertions or deletions as well as aminoacid substitutions. Such substitutions may be conservative, i.e. anamino acid residue is replaced with a chemically similar amino acidresidue. Examples of conservative substitutions are the replacementsamong the members of the following groups: 1) alanine, serine, andthreonine; 2) aspartic acid and glutamic acid; 3) asparagine andglutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine,and valine; and 6) phenylalanine, tyrosine, and tryptophan. One theother hand, it is also possible to introduce non-conservativealterations in the amino acid sequence.

Such modifications of the amino acid sequence include directedmutagenesis of single amino acid positions in order to simplifysub-cloning of the mutated lipocalin gene or its parts by incorporatingcleavage sites for certain restriction enzymes. In addition, thesemutations can also be incorporated to further improve the affinity of alipocalin mutein for a given target (cf. Examples 17-19 and 24). In oneembodiment, a mutation is introduced in at least one of the sequencepositions (of the lipocalin framework) that correspond to sequencepositions 21, 50, 51 and 83 of the linear polypeptide sequence of humantear lipocalin. Furthermore, mutations can be introduced in order tomodulate certain characteristics of the mutein such as to improvefolding stability or water solubility or to reduce aggregation tendency,if necessary.

The lipocalin muteins of the invention are able to bind the desiredtarget with detectable affinity, i.e. with an affinity constant ofpreferably at least 10⁵ M⁻¹. Lower affinities are generally no longermeasurable with common methods such as ELISA and therefore of secondaryimportance. Especially preferred are lipocalin muteins, which bind thedesired target with an affinity of at least 10⁶ M⁻¹, corresponding to adissociation constant of the complex of 1 μM. The binding affinity of amutein to the desired target can be measured by a multitude of methodssuch as fluorescence titration, competition ELISA or surface plasmonresonance.

It is clear to the skilled person that complex formation is dependent onmany factors such as concentration of the binding partners, the presenceof competitors, ionic strength of the buffer system etc. Selection andenrichment is generally performed under conditions allowing theisolation of lipocalin muteins having an affinity constant of at least10⁵ M⁻¹ to the target.

However, the washing and elution steps can be carried out under varyingstringency. A selection with respect to the kinetic characteristics ispossible as well. For example, the selection can be performed underconditions, which favor complex formation of the target with muteinsthat show a slow dissociation from the target, or in other words a lowk_(off) rate.

A tear lipocalin mutein of the invention may be used for complexformation with a given target. The target may be a non-naturaltarget/ligand. The target (ligand) may be any chemical compound in freeor conjugated form which exhibits features of an immunological hapten, ahormone such as steroid hormones or any biopolymer or fragment thereof,for example, a protein or protein domain, a peptide, anoligodeoxynucleotide, a nucleic acid, an oligo- or polysaccharide orconjugates thereof. In a preferred embodiment of the invention thetarget is a protein. The protein can be any globular soluble protein ora receptor protein, for example, a trans-membrane protein involved incell signaling, a component of the immune systems such as an MHCmolecule or cell surface receptor that is indicative of a specificdisease. The mutein may also be able to bind only fragments of aprotein. For example, a mutein can bind to a domain of a cell surfacereceptor, when it is part of the receptor anchored in the cell membraneas well as to the same domain in solution, if this domain can beproduced as a soluble protein as well. However the invention is by nomeans limited to muteins that only bind such macromolecular targets. Butit is also possible to obtain muteins of tear lipocalin by means ofmutagenesis which show specific binding affinity to ligands of low(er)molecular weight such as biotin, fluorescein or digoxigenin.

A tear lipocalin mutein of the invention typically exists as monomericprotein. However, it is also possible that an inventive lipocalin muteinis able to spontaneously dimerise or oligomerise. Although the use oflipocalin muteins that form stable monomers is usually preferred due tothe simplified handling of the protein, for example, the use oflipocalin muteins that form stable homodimers or multimers can even bepreferred here since such multimers can provide for a (further)increased affinity and/or avidity to a given target. Furthermore,oligomeric forms of the lipocalin mutein may have prolonged serumhalf-life.

For some applications, it is useful to employ the muteins of theinvention in a labeled form. Accordingly, the invention is also directedto lipocalin muteins which are conjugated to a label selected from thegroup consisting of enzyme labels, radioactive labels, colored labels,fluorescent labels, chromogenic labels, luminescent labels, haptens,digoxigenin, biotin, metal complexes, metals, and colloidal gold. Themutein may also be conjugated to an organic molecule. The term “organicmolecule” as used herein preferably denotes an organic moleculecomprising at least two carbon atoms, but preferably not more than sevenrotatable carbon bonds, having a molecular weight in the range between100 and 2000 Dalton, preferably 1000 Dalton, and optionally includingone or two metal atoms.

In general, it is possible to label the lipocalin mutein with anyappropriate chemical substance or enzyme, which directly or indirectlygenerates a detectable compound or signal in a chemical, physical orenzymatic reaction. An example for a physical reaction is the emissionof fluorescence upon irradiation or the emission of X-rays when using aradioactive label. Alkaline phosphatase, horseradish peroxidase orβ-galactosidase are examples of enzyme labels which catalyze theformation of chromogenic reaction products. In general, all labelscommonly used for antibodies (except those exclusively used with thesugar moiety in the Fc part of immunoglobulins) can also be used forconjugation to the muteins of the present invention. The muteins of theinvention may also be conjugated with any suitable therapeuticallyactive agent, e.g., for the targeted delivery of such agents to a givencell, tissue or organ or for the selective targeting of cells, e.g., oftumor cells without affecting the surrounding normal cells. Examples ofsuch therapeutically active agents include radionuclides, toxins, smallorganic molecules, and therapeutic peptides (such as peptides acting asagonists/antagonists of a cell surface receptor or peptides competingfor a protein binding site on a given cellular target). The lipocalinmuteins of the invention may, however, also be conjugated withtherapeutically active nucleic acids such as antisense nucleic acidmolecules, small interfering RNAs, micro RNAs or ribozymes. Suchconjugates can be produced by methods well known in the art.

For several applications of the muteins disclosed herein it may beadvantageous to use them in the form of fusion proteins. In preferredembodiments, the inventive lipocalin mutein is fused at its N-terminusor its C-terminus to a protein, a protein domain or a peptide such as asignal sequence and/or an affinity tag.

The fusion partner may confer new characteristics to the inventivelipocalin mutein such as enzymatic activity or binding affinity forother molecules. Examples of suitable fusion proteins are alkalinephosphatase, horseradish peroxidase, gluthation-5-transferase, thealbumin-binding domain of protein G, protein A, antibody fragments,oligomerization domains, lipocalin muteins of same or different bindingspecificity (which results in the formation of “duocalins”, cf.Schlehuber, S., and Skerra, A. (2001), Biol. Chem. 382, 1335-1342), ortoxins. In particular, it may be possible to fuse a lipocalin mutein ofthe invention with a separate enzyme active site such that both“components” of the resulting fusion protein together act on a giventherapeutic target. The binding domain of the lipocalin mutein attachesto the disease-causing target, allowing the enzyme domain to abolish thebiological function of the target. If two bispecific lipocalin muteinsof the inventions (i.e. each of them has two binding sites) are combinedinto a “duocalin”, a tetravalent molecule is formed. If for example aduocalin is generated from only one mutein having two binding sites thatspecifically bind biotin, a tetravalent molecule (homodimer) comparableto streptavidin (which is a homotetramer, in which each monomer bindsone biotin molecule) can be obtained. Due to expected avidity effectssuch a mutein might be a useful analytical tool in methods that make useof the detection of biotin groups. A lipocalin mutein that spontaneouslyforms homodimers or -multimers can, of course, also be used for such apurpose.

Affinity tags such as the Strep-tag® or Strep-tag® II (Schmidt, T. G. M.et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag,the His₆-tag or the HA-tag or proteins such as glutathione-S-transferasealso allow easy detection and/or purification of recombinant proteinsare further examples of preferred fusion partners. Finally, proteinswith chromogenic or fluorescent properties such as the green fluorescentprotein (GFP) or the yellow fluorescent protein (YFP) are suitablefusion partners for a lipocalin mutein of the invention as well.

The term “fusion protein” as used herein also comprises lipocalinmuteins according to the invention containing a signal sequence. Signalsequences at the N-terminus of a polypeptide direct this polypeptide toa specific cellular compartment, for example the periplasm of E. coli orthe endoplasmatic reticulum of eukaryotic cells. A large number ofsignal sequences is known in the art. A preferred signal sequence forsecretion a polypeptide into the periplasm of E. coli is the OmpA-signalsequence.

The present invention also relates to nucleic acid molecules (DNA andRNA) comprising nucleotide sequences coding for muteins as describedherein. Since the degeneracy of the genetic code permits substitutionsof certain codons by other codons specifying the same amino acid, theinvention is not limited to a specific nucleic acid molecule encoding afusion protein of the invention but includes all nucleic acid moleculescomprising nucleotide sequences encoding a functional fusion protein.

In one preferred embodiment of the nucleic acid molecule of inventionits sequence is derived from the coding sequence of human tearlipocalin. In other preferred embodiments the nucleic acid is derivedfrom the VEGP protein, VEG protein 2, LCN 1 or ALL 1 protein

In another preferred embodiment the nucleic acid sequence encoding amutein according to the invention comprises mutations at any two or moreof the sequence positions in the peptide segments corresponding to thesequence positions 7-14, 43-49, 70-77, and 87-97 of the linearpolypeptide sequence of human tear lipocalin with the sequence positionscorresponding to the positions 8, 9, 10, 11, 12, 13, 43, 45, 47, 70, 72,74, 75, 90, 92, 94, and 97 of human tear lipocalin being particularlypreferred.

In a further preferred embodiment the nucleic acid sequence encoding amutein according to the invention comprises mutations at any two or moreof the sequence positions in the peptide segments corresponding to thesequence positions 24-36, 53-66, 79-84, and 103-110 of the linearpolypeptide sequence of human tear lipocalin.

Also preferred are nucleic acid molecules encoding a mutein of theinvention comprising mutations at any two or more of the sequencepositions in the peptide segments corresponding to the sequencepositions 7-14, 43-49, 70-77, and 87-97 of the linear polypeptidesequence of human tear lipocalin mutations and additional mutations atany two or more of the sequence positions in the peptide segmentscorresponding to the sequence positions 24-36, 53-66, 79-84, and 103-110of the linear polypeptide sequence of human tear lipocalin.

The invention as disclosed herein also includes nucleic acid moleculesencoding TLPC muteins, which comprise additional mutations outside thesegments of experimental mutagenesis. Such mutations are often toleratedor can even prove to be advantageous, for example if they contribute toan improved folding efficiency, protein stability or ligand bindingaffinity of the mutein.

A nucleic acid molecule disclosed in this application may be “operablylinked” to a regulatory sequence (or regulatory sequences) to allowexpression of this nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or capable “to allow expression of anucleotide sequence” if it comprises sequence elements which containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions comprise a promoter which, inprokaryotes, contains both the promoter per se, i.e. DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′non-codingsequences involved in initiation of transcription and translation, suchas the −35/−10 boxes and the Shine-Dalgarno element in prokaryotes orthe TATA box, CAAT sequences, and 5′-capping elements in eukaryotes.These regions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the invention can include aregulatory sequence, preferably a promoter sequence. In anotherpreferred embodiment, a nucleic acid molecule of the invention comprisesa promoter sequence and a transcriptional termination sequence. Suitableprokaryotic promoters are, for example, the tet promoter, the lacUV5promoter or the T7 promoter. Examples of promoters useful for expressionin eukaryotic cells are the SV40 promoter or the CMV promoter.

The nucleic acid molecules of the invention can also be comprised in avector or any other cloning vehicles, such as plasmids, phagemids,phage, baculovirus, cosmids or artificial chromosomes. In a preferredembodiment, the nucleic acid molecule is comprised in a phasmid. Aphasmid vector denotes a vector encoding the intergenic region of atemperent phage, such as M13 or f1, or a functional part thereof fusedto the cDNA of interest. After superinfection of the bacterial hostcells with such an phagemid vector and an appropriate helper phage (e.g.M13K07, VCS-M13 or R408) intact phage particles are produced, therebyenabling physical coupling of the encoded heterologous cDNA to itscorresponding polypeptide displayed on the phage surface (reviewed,e.g., in Kay, B. K. et al. (1996) Phage Display of Peptides andProteins—A Laboratory Manual, 1st Ed., Academic Press, New York N.Y.;Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424, orRodi, D. J., and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93).

Such cloning vehicles can include, aside from the regulatory sequencesdescribed above and a nucleic acid sequence encoding a lipocalin muteinof the invention, replication and control sequences derived from aspecies compatible with the host cell that is used for expression aswell as selection markers conferring a selectable phenotype ontransformed or transfected cells. Large numbers of suitable cloningvectors are known in the art, and are commercially available.

The DNA molecule encoding lipocalin muteins of the invention, and inparticular a cloning vector containing the coding sequence of such alipocalin mutein can be transformed into a host cell capable ofexpressing the gene. Transformation can be performed using standardtechniques (Sambrook, J. et al. (1989), supra). Thus, the invention isalso directed to a host cell containing a nucleic acid molecule asdisclosed herein.

The transformed host cells are cultured under conditions suitable forexpression of the nucleotide sequence encoding a fusion protein of theinvention. Suitable host cells can be prokaryotic, such as Escherichiacoli (E. coli) or Bacillus subtilis, or eukaryotic, such asSaccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells,immortalized mammalian cell lines (e.g. HeLa cells or CHO cells) orprimary mammalian cells.

The invention also relates to a method for the generation of a muteinaccording to the invention or a fusion protein thereof, comprising:

-   -   (a) subjecting a nucleic acid molecule encoding a tear lipocalin        or a homologue thereof, wherein said tear lipocalin or homologue        thereof has at least 60% sequence homology with human tear        lipocalin, to mutagenesis at two or more different codons,        resulting in one or more mutein nucleic acid molecules(s);    -   (b) expressing the one or more mutein nucleic acid molecule(s)        obtained in (a) in a suitable expression system, and    -   (c) enriching at least one mutein having a detectable binding        affinity for a given target by means of selection and/or        isolation.

In further embodiments of this method, the nucleic acid molecule can beindividually subjected to mutagenesis at two or more different codons(i.e., usually nucleotide triplets) in any one, two, three or all fourabove-mentioned peptide segments arranged at either end of the β-barrelstructure. Accordingly, it is sufficient to exchange only one base in acodon if this exchange results in a change of the encoded amino acid.

In the method of generation a mutein or a fusion protein thereof isobtained starting from the nucleic acid encoding tear lipocalin or ahomologue thereof, which is subjected to mutagenesis and introduced intoa suitable bacterial or eukaryotic host organism by means of recombinantDNA technology (as already outlined above).

The coding sequence of, for example, human tear lipocalin (Redl, B. etal. (1992) J. Biol. Chem. 267, 20282-20287) can serve as a startingpoint for mutagenesis of the peptide segments selected in the presentinvention. For the mutagenesis of the amino acids in the N-terminalpeptide stretch and the three peptide loops BC, DE, and FG at the end ofthe β-barrel structure that is located opposite to the natural lipocalinbinding pocket as well as the four peptide loops AB, CD, EF, and GHencompassing said binding pocket, the person skilled in the art has athis disposal the various established standard methods for site-directedmutagenesis (Sambrook, J. et al. (1989), supra). A commonly usedtechnique is the introduction of mutations by means of PCR (polymerasechain reaction) using mixtures of synthetic oligonucleotides, which beara degenerate base composition at the desired sequence positions. The useof nucleotide building blocks with reduced base pair specificity, as forexample inosine, is another option for the introduction of mutationsinto a chosen sequence segment. A further possibility is the so-calledtriplet-mutagenesis. This method uses mixtures of different nucleotidetriplets each of which codes for one amino acid for the incorporationinto the coding sequence.

One possible strategy for introducing mutations in the selected regionsof the respective polypeptides is based on the use of fouroligonucleotides, each of which is partially derived from one of thecorresponding sequence segments to be mutated (cf. FIG. 3). Whensynthesizing these oligonucleotides, a person skilled in the art canemploy mixtures of nucleic acid building blocks for the synthesis ofthose nucleotide triplets which correspond to the amino acid positionsto be mutated so that codons encoding all natural amino acids randomlyarise, which at last results in the generation of a lipocalin peptidelibrary. For example, the first oligonucleotide corresponds in itssequence—apart from the mutated positions—to the coding strand for thepeptide segment to be mutated at the most N-terminal position of thelipocalin polypeptide. Accordingly, the second oligonucleotidecorresponds to the non-coding strand for the second sequence segmentfollowing in the polypeptide sequence. The third oligonucleotidecorresponds in turn to the coding strand for the corresponding thirdsequence segment. Finally, the fourth oligonucleotide corresponds to thenon-coding strand for the fourth sequence segment. A polymerase chainreaction can be performed with the respective first and secondoligonucleotide and separately, if necessary, with the respective thirdand fourth oligonucleotide.

The amplification products of both of these reactions can be combined byvarious known methods into a single nucleic acid comprising the sequencefrom the first to the fourth sequence segments, in which mutations havebeen introduced at the selected positions. To this end, both of theproducts can for example be subjected to a new polymerase chain reactionusing flanking oligonucleotides as well as one or more mediator nucleicacid molecules, which contribute the sequence between the second and thethird sequence segment. This procedure is schematically reproduced inFIG. 3. In the choice of the number and arrangement within the sequenceof the oligonucleotides used for the mutagenesis, the person skilled inthe art has numerous alternatives at his disposal.

The nucleic acid molecules defined above can be connected by ligationwith the missing 5′- and 3′-sequences of a nucleic acid encoding alipocalin polypeptide and/or the vector, and can be cloned in a knownhost organism. A multitude of established procedures are available forligation and cloning (Sambrook, J. et al. (1989), supra). For example,recognition sequences for restriction endonucleases also present in thesequence of the cloning vector can be engineered into the sequence ofthe synthetic oligonucleotides. Thus, after amplification of therespective PCR product and enzymatic cleavage the resulting fragment canbe easily cloned using the corresponding recognition sequences.

Longer sequence segments within the gene coding for the protein selectedfor mutagenesis can also be subjected to random mutagenesis via knownmethods, for example by use of the polymerase chain reaction underconditions of increased error rate, by chemical mutagenesis or by usingbacterial mutator strains. Such methods can also be used for furtheroptimization of the target affinity or specificity of a lipocalinmutein. Mutations possibly occurring outside the segments ofexperimental mutagenesis are often tolerated or can even prove to beadvantageous, for example if they contribute to an improved foldingefficiency or folding stability of the lipocalin mutein.

After expression of the nucleic acid sequences that were subjected tomutagenesis in an appropriate host, the clones carrying the geneticinformation for the plurality of respective lipocalin muteins, whichbind a given target can be selected from the library obtained. Wellknown techniques can be employed for the selection of these clones, suchas phage display (reviewed in Kay, B. K. et al. (1996) supra; Lowman, H.B. (1997) supra or Rodi, D. J., and Makowski, L. (1999) supra), colonyscreening (reviewed in Pini, A. et al. (2002) Comb. Chem. HighThroughput Screen. 5, 503-510), ribosome display (reviewed in Amstutz,P. et al. (2001) Curr. Opin. Biotechnol. 12, 400-405) or mRNA display asreported in Wilson, D. S. et al. (2001) Proc. Natl. Acad. Sci. USA 98,3750-3755.

An embodiment of the phage display technique (reviewed in Kay, B. K. etal. (1996), supra; Lowman, H. B. (1997) supra or Rodi, D. J., andMakowski, L. (1999), supra) using temperent M13 phage is given as anexample of a selection method according to the invention. However, it isnoted that other temperent phage such as f1 or lytic phage such as T7may be employed as well. For the exemplary selection method, M13phagemids (cf. also above) are produced which allow the expression ofthe mutated lipocalin nucleic acid sequence as a fusion protein with asignal sequence at the N-terminus, preferably the OmpA-signal sequence,and with the capsid protein pIII of the phage M13 or fragments thereofcapable of being incorporated into the phage capsid at the C-terminus.The C-terminal fragment ΔpIII of the phage capsid protein comprisingamino acids 217 to 406 of the wild type sequence is preferably used toproduce the fusion proteins. Especially preferred is a C-terminalfragment of pIII, in which the cysteine residue at position 201 ismissing or is replaced by another amino acid.

The fusion protein may comprise additional components such as anaffinity tag, which allows the immobilization and/or purification of thefusion protein or its parts. Furthermore, a stop codon can be locatedbetween the sequence regions encoding the lipocalin or its muteins andthe phage capsid gene or fragments thereof, wherein the stop codon,preferably an amber stop codon, is at least partially translated into anamino acid during translation in a suitable suppressor strain.

For example, the phagemid vector pTLPC7 (FIG. 4) can be used for theconstruction of a phage library encoding human tear lipocalin muteins.The inventive nucleic acid molecules coding for the mutated peptidesegments are inserted into the vector using the BstXI restriction sites.Recombinant vectors are then transformed into a suitable host strainsuch as E. coli XL1-Blue. The resulting library is subsequentlysuperinfected in liquid culture with an appropriate M13-helper phage inorder to produce functional phage. The recombinant phagemid displays thelipocalin mutein on its surface as a fusion with the coat protein pIIIor a fragment thereof, while the N-terminal signal sequence of thefusion protein is normally cleaved off. On the other hand, it also bearsone or more copies of the native capsid protein pIII supplied by thehelper phage and is thus capable of infecting a recipient, in general abacterial strain carrying a F- or F′-plasmid. During or after infectiongene expression of the fusion protein comprised of the lipocalin muteinand the capsid protein pIII can be induced, for example by addition ofanhydrotetracycline. The induction conditions are chosen such that asubstantial fraction of the phage obtained displays at least onelipocalin mutein on their surface. Various methods are known forisolating the phage, such as precipitation with polyethylene glycol.Isolation typically occurs after an incubation period of 6-8 hours.

The isolated phage are then subjected to a selection process byincubating them with a given target, wherein the target is present in aform allowing at least a temporary immobilization of those phagedisplaying muteins with the desired binding activity. Severalimmobilization methods are known in the art. For example, the target canbe conjugated with a carrier protein such as serum albumin and be boundvia this carrier to a protein-binding surface such as polystyrene.Microtiter plates suitable for ELISA techniques or so-called“immunosticks” are preferred. Alternatively, conjugates of the targetcan also be implemented with other binding groups such as biotin. Thetarget can then be immobilized on surfaces, which will selectively bindthis group, such as microtiter plates or paramagnetic particles coatedwith avidin or streptavidin.

For example, the phage particles are captured by binding to therespective target immobilized on the surface. Unbound phage particlesare subsequently removed by iterative washing. For the elution of boundphage, free target (ligand) molecules can be added to the samples as acompetitor. Alternatively, elution can also be achieved by addingproteases or under moderately denaturing conditions, e.g. in thepresence of acids, bases, detergents or chaotropic salts. A preferredmethod is the elution using buffers having pH 2.2, followed byneutralization of the solution. The eluted phage may then be subjectedto another selection cycle. Preferably, selection is continued until atleast 0.1% of the clones comprise lipocalin muteins with detectableaffinity for the respective target. Depending on the complexity of thelibrary employed 2-8 cycles are required to this end.

For the functional analysis of the selected lipocalin muteins, an E.coli host strain is infected with the phagemids obtained and phagemidDNA is isolated using standard techniques (Sambrook, J. et al. (1989),supra). The mutated sequence fragment or the entire lipocalin muteinnucleic acid sequence can be sub-cloned in any suitable expressionvector. The recombinant lipocalin muteins obtained can be purified fromtheir host organism or from a cell lysate by various methods known inthe art such as gel filtration or affinity chromatography.

However, the selection of lipocalin muteins can also be performed usingother methods well known in the art. Furthermore, it is possible tocombine different procedures. For example, clones selected or at leastenriched by phage display can subsequently be subjected to acolony-screening assay in order to directly isolate a particularlipocalin mutein with detectable binding affinity for a given target.Additionally, instead of generating a single phage library comparablemethods can be applied in order to optimize a mutein with respect to itsaffinity or specificity for the desired target by repeated, optionallylimited mutagenesis of its coding nucleic acid sequence.

Once a mutein with affinity to a given target have been selected, it isadditionally possible to subject such a mutein to further mutagenesis inorder to select variants of even higher affinity from the new librarythus obtained. The affinity muturation can be achieved by site specificmutation based on rational design or a random mutation One possibleapproach for affinity maturation is the use of error-prone PCR, whichresults in point mutations over a selected range of sequence positionsof the lipocalin mutein (cf. Example 17). The error prone PCR can becarried out in accordance with any known protocol such as the onedescribed by Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603. Othermethods of random mutagenesis that are suitable for affinity maturationinclude random insertion/deletion (RID) mutagenesis as described byMurakami, H et al. (2002) Nat. Biotechnol. 20, 76-81 or nonhomologousrandom recombination (NRR) as described by Bittker, J. A et al. (2002)Nat. Biotechnol. 20, 1024-1029. Affinity maturation can also be carriedout according to the procedure described in WO 00/5308 or Schlehuber, S.et al. (2000) J. Mol. Biol. 297, 1105-1120, where muteins of thebilin-binding protein having high affinity to digoxigenin were obtained.

The invention also relates to a method for the production of a mutein ofthe invention, wherein the mutein, a fragment of the mutein or a fusionprotein of the mutein and another polypeptide is produced starting fromthe nucleic acid coding for the mutein by means of genetic engineeringmethods. The method can be carried out in vivo, the mutein can forexample be produced in a bacterial or eucaryotic host organism and thenisolated from this host organism or its culture. It is also possible toproduce a protein in vitro, for example by use of an in vitrotranslation system.

When producing the mutein in vivo a nucleic acid encoding a mutein ofthe invention is introduced into a suitable bacterial or eukaryotic hostorganism by means of recombinant DNA technology (as already outlinedabove). For this purpose, the host cell is first transformed with acloning vector comprising a nucleic acid molecule encoding a mutein ofthe invention using established standard methods (Sambrook, J. et al.(1989), supra). The host cell is then cultured under conditions, whichallow expression of the heterologous DNA and thus the synthesis of thecorresponding polypeptide. Subsequently, the polypeptide is recoveredeither from the cell or from the cultivation medium. Since manylipocalins comprise intramolecular disulfide bonds, it can be preferredto direct the polypeptide to a cell compartment having an oxidizingredox-milieu using an appropriate signal sequence. Such an oxidizingenvironment is provided in the periplasm of Gram-negative bacteria suchas E. coli or in the lumen of the endoplasmatic reticulum of eukaryoticcells and usually favors the correct formation of the disulfide bonds.It is, however, also possible to generate a mutein of the invention inthe cytosol of a host cell, preferably E. coli. In this case, thepolypeptide can, for instance, be produced in form of inclusion bodies,followed by renaturation in vitro. A further option is the use ofspecific host strains having an oxidizing intracellular milieu, whichthus allow the production of the native protein in the cytosol.

However, a mutein of the invention may not necessarily be generated orproduced only by use of genetic engineering. Rather, a lipocalin muteincan also be obtained by chemical synthesis such as Merrifield solidphase polypeptide synthesis. It is for example possible that promisingmutations are identified using molecular modeling and then to synthesizethe wanted (designed) polypeptide in vitro and investigate the bindingactivity for a given target. Methods for the solid phase and/or solutionphase synthesis of proteins are well known in the art (reviewed, e.g.,in Lloyd-Williams, P. et al. (1997) Chemical Approaches to the Synthesisof Peptides and Proteins. CRC Press, Boca Raton, Fields, G. B., andColowick, S. P. (1997) Solid-Phase Peptide Synthesis. Academic Press,San Diego, or Bruckdorfer, T. et al. (2004) Curr. Pharm. Biotechnol. 5,29-43).

The invention also relates to a pharmaceutical composition comprising atleast one inventive mutein or a fusion protein thereof and apharmaceutically acceptable excipient.

The lipocalin muteins according to the invention can be administered viaany parenteral or non-parenteral (enteral) route that is therapeuticallyeffective for proteinaceous drugs. Parenteral application methodscomprise, for example, intracutaneous, subcutaneous, intramuscular orintravenous injection and infusion techniques, e.g. in the form ofinjection solutions, infusion solutions or tinctures, as well as aerosolinstallation and inhalation, e.g. in the form of aerosol mixtures,sprays or powders. Non-parenteral delivery modes are, for instance,orally, e.g. in the form of pills, tablets, capsules, solutions orsuspensions, or rectally, e.g. in the form of suppositories. The muteinsof the invention can be administered systemically or topically informulations containing conventional non-toxic pharmaceuticallyacceptable excipients or carriers, additives and vehicles as desired.

In a preferred embodiment of the present invention the pharmaceutical isadministered parenterally to a mammal, and in particular to humans, withaerosol installation being one of the most preferable applicationmethod, taking advantage of the low molecular weight of the muteins.

Accordingly, the muteins of the present invention can be formulated intocompositions using pharmaceutically acceptable ingredients as well asestablished methods of preparation (Gennaro, A. L. and Gennaro, A. R.(2000) Remington: The Science and Practice of Pharmacy, 20th Ed.,Lippincott Williams & Wilkins, Philadelphia, Pa.). To prepare thepharmaceutical compositions, pharmaceutically inert inorganic or organicexcipients can be used. To prepare e.g. pills, powders, gelatin capsulesor suppositories, for example, lactose, talc, stearic acid and itssalts, fats, waxes, solid or liquid polyols, natural and hardened oils.Suitable excipients for the production of solutions, suspensions,emulsions, aerosol mixtures or powders for reconstitution into solutionsor aerosol mixtures prior to use include water, alcohols, glycerol,polyols, and suitable mixtures thereof as well as vegetable oils.

The pharmaceutical composition may also contain additives, such as, forexample, fillers, binders, wetting agents, glidants, stabilizers,preservatives, emulsifiers, and furthermore solvents or solubilizers oragents for achieving a depot effect. The latter is that fusion proteinsmay be incorporated into slow or sustained release or targeted deliverysystems, such as liposomes and microcapsules.

The formulations can be sterilized by numerous means, includingfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile medium justprior to use.

As is evident from the above disclosure, a mutein of the presentinvention or a fusion protein or a conjugate thereof can be employed inmany applications. In general, such a mutein can be used in allapplications antibodies are used, except those with specifically rely onthe glycosylation of the Fc part.

A mutein of the invention can also be used for the targeting of acompound to a preselected site. For such a purpose the mutein iscontacted with the compound of interest in order to allow complexformation. Then the complex comprising the mutein and the compound ofinterest are delivered the preselected site. This use is in particularsuitable, but not restricted to, for delivering a drug (selectively) tothe site such an infected body part or organ which is supposed to betreated with the drug.

Another use of the inventive muteins is the binding/detection of a giventarget or target molecule, comprising contacting the mutein with a testsample supposed to contain said target, and detecting of themutein/target complex by a suitable signal. A mutein can also be usedfor the separation of a given target, comprising contacting the muteinwith a sample supposed to contain said target in order to allow complexformation, and separating the mutein/target complex from the sample. Insuch uses the complex comprising the mutein and the target may beimmobilized on any suitable solid phase.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is plasmon surface resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The muteins disclosed herein and its derivatives can thus be used inmany fields similar to antibodies or fragments thereof. In addition totheir use for binding to a support, allowing the target of a givenmutein or a conjugate or a fusion protein of this target to beimmobilized or separated, the muteins can be used for labeling with anenzyme, an antibody, a radioactive substance or any other group havingbiochemical activity or defined binding characteristics. By so doing,their respective targets or conjugates or fusion proteins thereof can bedetected or brought in contact with them. For example, muteins of theinvention can serve to detect chemical structures by means ofestablished analytical methods (e.g. ELISA or Western Blot) or bymicroscopy or immunosensorics. Here, the detection signal can either begenerated directly by use of a suitable mutein conjugate or fusionprotein or indirectly by immunochemical detection of the bound muteinvia an antibody.

Numerous possible applications for the inventive muteins also exist inmedicine. In addition to their use in diagnostics and drug delivery, amutant polypeptide of the invention, which binds, for example, tissue-or tumor-specific cellular surface molecules can be generated. Such amutein may, for example, be employed in conjugated form or as a fusionprotein for “tumor imaging” or directly for cancer therapy.

Another related and preferred use of a mutein described herein is targetvalidation, i.e. the analysis whether a polypeptide assumed to beinvolved in the development or progress of a disease or disorder isindeed somehow causative of that disease or disorder. This use forvalidating a protein as a pharmacological drug target takes advantage ofthe ability of a mutein of the present invention to specificallyrecognize a surface area of a protein in its native conformation, i.e.to bind to a native epitope. In this respect, it is to be noted thatthis ability has been reported only for a limited number of recombinantantibodies. However, the use of an inventive mutein for validation of adrug target is not limited to the detection of proteins as targets, butalso includes the detection of protein domains, peptides, nucleic acidmolecules, organic molecules or metal complexes.

The invention is further illustrated by the following Figures andExamples, in which:

FIG. 1 shows the polypeptide sequence of mature human tear lipocalin(SWISS-PROT Data Bank Accession Number M90424).

FIG. 2 schematically depicts the structure of the lipocalin fold.

FIG. 3 schematically illustrates the generation of the library of tearlipocalin muteins randomized at the closed end of the β-barrel at thenucleic acid level.

FIG. 4 schematically depicts the phagemid vector pTLPC7.

FIG. 5 schematically depicts the phasmid vector pTLPC6.

FIG. 6 schematically illustrates the generation of the library of tearlipocalin muteins randomized at the open end of the 3-barrel at thenucleic acid level.

FIG. 7 schematically depicts the phagemid vector pTLPC12.

FIG. 8 shows a schematic drawing of the expression vector pTLPC8.

FIG. 9 depicts the binding of the TLPC mutein S69.4 O13 to rhuVEGF165 inan ELISA.

FIG. 10 depicts the binding of the TLPC mutein S76.1H10 Monomer to hCD22in an ELISA.

FIG. 11 depicts the binding of the TLPC mutein S76.1 H10 Dimer to hCD22in an ELISA.

FIG. 12 depicts the binding of the TLPC mutein S67.7 C6 to human CD25 inan ELISA.

FIG. 13 schematically depicts the mammalian transfection vectorCD25-pcDNA3.1Zeo(+).

FIG. 14 shows the staining of CHO cells expressing human CD25 withfluorescein-labeled TLPC mutein S67.7 C6.

FIG. 15 schematically depicts the expression vector pTLPC9.

FIG. 16 depicts the binding of the monomeric fraction of the TLPC muteinF92.8 M1.2 E15 to human CD25 in an ELISA.

FIG. 17 depicts the binding of the dimeric fraction of the TLPC muteinF92.8 M1.2E15 to human CD25 in an ELISA.

FIG. 18 schematically depicts the mammalian transfection vectorCD154-pcDNA3.1Zeo(+).

FIG. 19 shows the staining of CHO cells expressing human CD25 withfluorescein-labeled TLPC mutein F92.8 M1.2 E15;

FIG. 20 depicts the binding of the TLPC muteins S99.3H24, S99.3 C13 andS99.4 F15, respectively to human CD25 in an ELISA.

FIG. 21 schematically depicts the expression vector pTLPC14.

FIG. 22 depicts the binding of the TLPC mutein S100.1 I08 monomer anddimer to hCD33-Fc in an ELISA.

FIG. 23 depicts the binding of the TLPC mutein S101.2 A20 to hCD33-Fc inan ELISA.

FIG. 24 depicts the binding of the TLPC mutein S101.2 008 monomer anddimer to hCD33-Fc in an ELISA.

FIG. 25 depicts the binding of the TLPC mutein S100.1 I08 Dimer tohCD33-Fc in BIAcore experiments.

FIG. 26 depicts the binding of the TLPC mutein S101.2 A20 to hCD33-Fc inBIAcore experiments.

FIG. 27 depicts the binding of the TLPC mutein S101.2 008 to hCD33-Fc inBIAcore.

FIG. 1 shows the polypeptide sequence of mature human tear lipocalin(SWISS-PROT Data Bank Accession Number M90424, 158 amino acids, cf. alsoRedl B. (2000) Biochim. Biophys. Acta, supra). In this respect, it isnoted that a human protein that was modified as follows was used in thefollowing examples for the generation of lipocalin muteins. First, thefirst four N-terminal amino acid residues of the deposited sequence ofhuman tear lipocalin (HHLL) were deleted. Second, the last twoC-terminal amino acid residues (SD) were also deleted. Third, the wildtype sequence at sequence positions 5 to 7 (ASD) was changed to GGD.These changes are reflected in the attached sequence listings, in whichthe amino acids GGD are indicated as first three residues of the usedtear lipocalin. The four segments (AB, CD, EF and GH) at the open end ofthe β-barrel in which amino acids are exchanged are marked below thesequence of TLPC by double underlining. The segments BC, DE and PG andthe N-terminal peptide stretch in which mutations are introduced tocreate a binding site at the closed end of the β-barrel are marked inbold and single underlining. The sequences positions of TLPC that aremutated in the examples are additionally labeled with asterisks.

FIG. 2 schematically illustrates the characteristic features of thelipocalin fold (according to Flower, D. R. (1996), supra). The eightβ-strands of the antiparallel β-sheet which form the β-barrel) are shownas arrows and labeled A to H (a ninth β-strand, designated I which isadditionally present in some lipocalins, is also schematically shown).The hydrogen-bonded connection of two strands is indicated by a pair ofdotted lines between them. The connecting loops are shown as solidcurved lines. The two ends of the β-barrel are topologically distinct.One end has four β-hairpins (loops AB, CD, EF and GH), the opening ofthe known ligand binding site of the lipocalins is here and called theopen end. The other end of the β-barrel has three loops (BC, DE and FG),which together with the N-terminal polypeptide region build the closedend and are used in the present invention to introduce an alternativebinding site. The parts which form the three main structurally conservedregions (SCRs) of the fold, SCR1, SCR2 and SCR3, are marked as boxes.

FIG. 3 schematically shows a strategy for the concerted mutagenesis of17 selected amino acid positions in the modified TLPC by repeatedpolymerase chain reaction (PCR). For the sequence near the N-terminusand for each of the three peptide loops BC, DE, and FG, respectively, inwhich the amino acids are to be mutated, an oligodeoxynucleotide wassynthesized, (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:6), bearing random nucleotides as indicated in the sequence listing. Dueto the composition chosen, from the altogether three possible stopcodons only the amber stop codon, TAG, was allowed at the mutatedcodons, which is translated as glutamine in the E. coli supE strainsXL1-blue (Bullock et al. (1987) BioTechniques 5, 376-378) or TG1(Sambrook et al., supra). For certain applications, for example geneexpression in other bacterial strains or organisms, such a nonsensecodon can be substituted by a glutamine-encoding codon, e.g., bysite-directed mutagenesis. A nucleic acid fragment with 159 base pairswas amplified (PCR No. 1, A) with the respective primers SEQ ID NO: 3and SEQ ID NO: 4 using the pTLPC6 plasmid-DNA (SEQ ID NO: 2) as atemplate. In another PCR, a nucleic acid fragment with 123 base pairswas amplified (PCR no. 1, B) with the primers SEQ ID NO: 5 and SEQ IDNO: 6, respectively, also using pTLPC6 as template. The mixture of bothPCR products served as a template in another amplification (PCR No. 2)with the two 5′-biotinylated flanking PCR primers, namely SEQ ID NO: 7and SEQ ID NO: 8, and a mediating primer SEQ ID NO: 9, resulting in theamplification of a DNA fragment of 341 base pairs. This fragmentcomprising a mixture of all 17 mutated codons was subsequently clonedinto the vector pTPLC7 using the two BstXI restriction sites, thespecial arrangement of which led to two non-compatible overhanging DNAends enabling a particularly efficient ligation. The ligation efficiencycould be improved by purification of the digested PCR-fragment byparamagnetic streptavidin coated beads. The amino acid substitutionGlu104Gln as well as the silent mutations in the codon for Ala-3 of theompA signal sequence, in the codon for Ala21 and His106 were previouslyaccomplished during the construction of pTLPC6 in order to introduceboth of the BstX1 restriction sites into the TLPC coding sequence.

FIG. 4 shows a schematic drawing of the vector pTLPC7 encoding a fusionprotein comprised of the OmpA signal sequence (OmpA), a modified TLPCwith the amino acid substitutions Ala5Asp, Ser6Gly, Asp7Gly, Cys101Ser,and Glu104Gln (for the TLPC cDNA, see Redl et al., supra) and atruncated form of the M13 coat protein pIII, comprising amino acids 217to 406 (pIII). Gene expression is under the control of the tetracyclinepromoter/operator (tet^(p/o)) system. Transcription is terminated at thelipoprotein transcription terminator (t_(lpp)). The vector furthercomprises an origin of replication (ori), the intergenic region of thefilamentous phage f1 (f1-IG), the ampicillin resistance gene (bla)coding for β-lactamase and the tetracycline repressor gene (tetR). Anamber stop codon, which is partially translated into Gln in SupE ambersuppressor host strain, is located between the TLPC coding region andthe coding region for the truncated phage coat protein pIII. Both theBstXI-restriction sites used for the cloning of the mutated genecassette and the restriction sites flanking the structural gene arelabeled. The nucleic acid sequence of a XbaI-HindIII segment of pTLPC7is shown together with the encoded amino acid sequence in the sequencelisting as SEQ ID NO: 1. The vector sequence outside this region isidentical with that of pASK75, the complete nucleotide sequence of whichis given in the German patent publication DE 44 17 598 A1.

FIG. 5 shows a schematic drawing of the vector pTLPC6. pTLPC6 encodes afusion protein comprised of the OmpA signal sequence, a modified TLPCaccording to FIG. 1, and the Strep-tag® II affinity tag. Otherwise, thevector is identical to pTLPC7. The nucleic acid sequence of aXbaI-HindIII segment of pTLPC6 is shown together with the encoded aminoacid sequence in the sequence listing as SEQ ID NO: 2. The vectorsequence outside this region is identical with that of pASK75, thecomplete nucleotide sequence of which is given in the German patentpublication DE44 17 598A1.

FIG. 6 schematically shows a strategy for the concerted mutagenesis of17 or 19 selected amino acid positions in the modified TLPC by repeatedpolymerase chain reaction (PCR). For randomization of loop AB threeforward oligodeoxynucleotides were synthesized (SEQ ID NO: 26, SEQ IDNO: 27, and SEQ ID NO: 28), which differs in length coding for arandomized loop AB as well as an extension by two and four amino acids,respectively) and one reverse oligodeoxynucleotide for loop CD (SEQ IDNO: 29). Furthermore a pair of two oligodeoxynucleotides was synthesized(SEQ ID NO: 30, and SEQ ID NO: 31) for the peptide loops EF and GH,respectively. These oligonucleotides are bearing random nucleotides asindicated in the sequence listing in which the amino acids are to bemutated. Three nucleic acid fragments with 142, 148, and 154 base pairswere amplified (PCR No. 1, A) with the respective primers SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29 using the pTLPC12plasmid-DNA (FIG. 7, SEQ ID NO: 23) as a template. In another PCR, anucleic acid fragment with 119 base pairs was amplified (PCR No. 1, B)with the primers SEQ ID NO: 30 and SEQ ID NO: 31, respectively, alsousing pTLPC12 as template. The mixture of PCR fragment B resulting fromPCR No. 1B with each of the three PCR fragments A resulting from PCR No.1A (varying in length of loop AB) served as templates in anotheramplification (PCR No. 2) employing the two 5′-biotinylated flanking PCRprimers given as SEQ ID NO: 33 and SEQ ID NO: 34 together with amediating primer SEQ ID NO: 32. This PCR resulted in an amplification ofDNA fragments consisting of 336, 342, and 348 base pairs in size whichcomprises nearly the whole structural gene of tear lipocalin with either17 (for loop AB and loop AB extended by 4 amino acids) or 19 (for loopAB extended by 2 amino acids) mutated codons. The fragments weresubsequently cloned into the vector pTPLC12 using the two BstXIrestriction sites, the special arrangement of which led to twonon-compatible overhanging DNA ends enabling a particularly efficientligation. The ligation efficiency could be improved by purification ofthe digested PCR-fragment by paramagnetic streptavidin coated beads. Theamino acid substitution Ser14Pro and Lys114Gln as well as the silentmutations in the codon for Met21, Val110, and in the codon for Val116were previously accomplished during the construction of pTLPC12 in orderto introduce both of the BstXI restriction sites into the TLPC codingsequence.

FIG. 7 shows a schematic drawing of the vector pTLPC12 encoding a fusionprotein comprised of the OmpA signal sequence (OmpA), a T7 detection tag(17), a modified TLPC with the amino acid substitutions Ser14Pro,Lys114Gln, Cys101Ser, and Glu104Gln (for the TLPC cDNA, see Redi et al.,supra) and a truncated form of the M13 coat protein pIII, comprisingamino acids 217 to 406 (pIII). Gene expression is under the control ofthe tetracycline promoter/operator (tet^(p/o)) system. Transcription isterminated at the lipoprotein transcription terminator (t_(lpp)). Thevector further comprises an origin of replication (ori), the intergenicregion of the filamentous phage f1 (f1-IG), the ampicillin resistancegene (bla) coding for β-lactamase and the tetracycline repressor gene(tetR). An amber stop codon, which is partially translated into Gln inSupE amber suppressor host strain, is located between the TLPC codingregion and the coding region for the truncated phage coat protein pIII.Both the BstXI-restriction sites used for the cloning of the mutatedgene cassette and the restriction sites flanking the structural gene arelabeled. The nucleic acid sequence of a XbaI-HindIII segment of pTLPC12is shown together with the encoded amino acid sequence in the sequencelisting as SEQ ID NO: 23. The vector sequence outside this region isidentical with that of pASK75, the complete nucleotide sequence of whichis given in the German patent publication DE 44 17 598 A1.

FIG. 8 shows a schematic drawing of the expression vector pTLPC8. pTLPC8codes for a fusion protein of the OmpA signal sequence with a modifiedtear lipocalin according to (FIG. 4) followed by the T7 detection tag(T7) and the C-terminal Strep-tag® II. A relevant segment of the nucleicacid sequence of pTLPC8 is reproduced together with the encoded aminoacid sequence in the sequence listing as SEQ ID NO: 24. The segmentbegins with the XbaI restriction site and ends with the HindIIIrestriction site. The vector elements outside this region are identicalwith the vector pASK75, the complete nucleotide sequence of which isexhibited in the German patent publication DE 44 17 598 A1.

FIG. 9 shows a graphical representation of the data from Example 6, inwhich binding measurements with TLPC mutein and the prescribed targetrhuVEGF165 as well as the unrelated target BSA were performed by ELISA.Binding of TLPC mutein S69.4 013 (filled circles) to rhuVEGF165immobilized on the ELISA plate was compared with the interaction of themuteins with BSA (open circles) as control (also immobilized on theELISA plate). The TLPC muteins bound rhuVEGF165 in aconcentration-dependent manner, whereas no significant binding signalsto the unrelated target (open circles) were detectable.

FIG. 10 shows a graphical representation of the data from Example 10, inwhich binding measurements of the TLPC mutein S76.1H10 Monomer with theprescribed target hCD22 as well as the unrelated targets hIgG1, HSA andhCD33-Fc were performed by ELISA. Binding of the immobilized TLPC muteinS76.1H10 Monomer to hCD22 (closed squares) was compared with theinteraction of the mutein with hIgG1 (open triangles), HSA (opencircles) and hCD33-Fc (open diamonds). The TLPC mutein binds hCD22 in aconcentration-dependent manner, whereas no significant binding signalswere detectable to the unrelated targets.

FIG. 11 shows a graphical representation of the data from Example 10, inwhich binding measurements of the TLPC mutein S76.1H10 Dimer with theprescribed target hCD22 as well as the unrelated targets hIgG1, HSA andhCD33-Fc were performed by ELISA. Binding of the immobilized TLPC muteinS76.1H10 Dimer to hCD22 (closed circles) was compared with theinteraction of the mutein with hIgG1 (open triangles), HSA (opensquares) and hCD33-Fc (open diamonds). The TLPC mutein binds hCD22 in aconcentration-dependent manner, whereas no significant binding signalswere detectable to the unrelated targets.

FIG. 12 shows a graphical representation of the data from Example 14, inwhich binding measurements with the TLPC mutein S67.7 C6 and theprescribed target CD25 as well as the unrelated targets capture mAb,HSA, FCS and captured human IgG Fc-fragment were performed by ELISA.Binding of the TLPC mutein S67.7 C6 (closed circle) to CD25-Fc(immobilized on the ELISA plate via a capture mAb) was compared with theinteraction of the mutein with capture mAb (open circle) as control(also immobilized on the ELISA plate). The TLPC mutein S67.7 C6 bindsCD25 in a concentration-dependent manner, whereas no significant bindingsignal to the unrelated target capture mAb (open symbol) was detectable.A control binding curve is only shown for this unrelated target, butsimilar results were obtained for the other control targets tested.

FIG. 13 shows a schematic drawing of the mammalian transfection vectorCD25-pcDNA3.1Zeo(+). This vector codes for the complete cDNA sequence ofhuman CD25 according to NCBI ACCESSION NM_(—)000417 [gi:4557666]. Arelevant segment of the nucleic acid sequence of human CD25 isreproduced together with the encoded amino acid sequence in the sequencelisting as SEQ ID NO: 10. The segment begins with a HindIII restrictionsite and ends with the XhoI restriction site. The vector elementsoutside this region are identical with those of the vectorpcDNA3.1Zeo(+) (Invitrogen).

FIG. 14 shows the staining of CHO cells expressing human CD25 withfluorescein labeled TLPC mutein S67.7 C6. CHO cells transfected with theexpression vector CD25-pcDNA3.1Zeo(+) (CHO-CD25; upper panels) or theparental vector pcDNA3.1Zeo(+) (CHO; lower panels) were incubated withthe CD25-specific mutein S67.7 C6 labeled with fluorescein at anequimolar ratio (left panels; histograms with solid lines) orFITC-labeled CD25-specific mAb (right panels; histograms with solidlines). In parallel, these cell lines were incubated with therecombinant wild type TLPC encoded by pTLPC8 labeled with fluorescein atequimolar ratio (left panels; histograms with broken lines) orFITC-labeled IgG1 (right panels; histograms with broken lines), both ascontrols. Both the CD25-specific mutein S67.7 C6 and the CD25-specificmAb show significant staining of the CHO cell line expressing human CD25while no significant staining of the mock-transfected CHO cell lineoccurs. The controls wild type TLPC and IgG1 show no significant bindingto both cell lines tested.

FIG. 15 shows a schematic drawing of pTLPC9. This vector codes for afusion protein of the OmpA signal sequence, a modified tear lipocalinaccording to FIG. 1, the Strep-tag® II and an albumin-binding domain(abd) of protein G from Streptococcus (Kraulis et al. (1996) FEBS Lett.378, 190-194). An amber stop codon has been introduced between theStrep-tag® II and the C-terminal albumin binding domain to allow solubleexpression of the TLPC mutein without the ABD when employing anon-supressor E. coli strain. A relevant segment of the nucleic acidsequence of pTLPC9 is reproduced together with the encoded amino acidsequence in the sequence listing as SEQ ID NO: 22. The segment beginswith an XbaI restriction site and ends with the HindIII restrictionsite. The vector elements outside this region are identical with thoseof the vector pASK75, the complete nucleotide sequence of which isexhibited in the German patent publication DE 44 17 598 A1.

FIG. 16 shows a graphical representation of the data from Example 21, inwhich binding measurements with the monomeric fraction of TLPC muteinF92.8 M1.2 E15 and the prescribed target CD25 as well as the unrelatedtargets capture mAb, HSA, FCS and captured human IgG Fc-fragment wereperformed by ELISA. Binding of the monomeric fraction of TLPC muteinF92.8 M1.2 E15 (closed circle) to CD25-Fc (immobilized on the ELISAplate via a capture mAb) was compared with the interaction of the muteinwith capture mAb (open circle) as control (also immobilized on the ELISAplate). The monomeric fraction of TLPC mutein F92.8 M1.2 E15 binds CD25in a concentration-dependent manner, whereas no significant bindingsignal to the unrelated target capture mAb (open symbol) was detectable.A control binding curve is only shown for this unrelated target, butsimilar results were obtained for the other control targets tested.

FIG. 17 shows a graphical representation of the data from Example 21, inwhich binding measurements with the dimeric fraction of TLPC muteinF92.8 M1.2 E15 and the prescribed target CD25 as well as the unrelatedtargets capture mAb, HSA, FCS and captured human IgG Fc fragment wereperformed by ELISA. Binding of the dimeric fraction of TLPC mutein F92.8M1.2 E15 (closed circle) to CD25-Fc (immobilized on the ELISA plate viaa capture mAb) was compared with the interaction of the mutein withcapture mAb (open circle) as control (also immobilized on the ELISAplate). The dimeric fraction of TLPC mutein F92.8 M1.2 E15 binds CD25 ina concentration-dependent manner, whereas no significant binding signalto the unrelated target capture mAb (open symbol) was detectable. Acontrol binding curve is only shown for this unrelated target, butsimilar results were obtained for the other control targets tested.

FIG. 18 shows a schematic drawing of the mammalian transfection vectorCD154-pcDNA3.1Zeo(+). This vector codes for the complete cDNA sequenceof human CD154 according to NCBI ACCESSION BC_(—)074950 [gi:49902361]. Arelevant segment of the nucleic acid sequence of human CD154 isreproduced together with the encoded amino acid sequence in the sequencelisting as SEQ ID NO: 11. The segment begins with a XhoI restrictionsite and ends with the ApaI restriction site. The vector elementsoutside this region are identical with those of the vectorpcDNA3.1Zeo(+) (Invitrogen).

FIG. 19 shows the staining of CHO cells expressing human CD25 withfluorescein labeled TLPC mutein F92.8 M1.2 E15. CHO cells transfectedwith the expression vector CD25-pcDNA3.1Zeo(+) (CHO-CD25; upper panels)or the expression vector CD154-pcDNA3.1Zeo(+) (CHO-CD154; lower panels)were incubated with the affinity-improved CD25-specific mutein F92.8M1.2 E15 labeled with fluorescein at a twofold molar ratio (left panels;histograms with solid lines) or FITC-labeled CD25-specific mAb (rightpanels; histograms with solid lines). In parallel, these cell lines wereincubated with the recombinant wild type TLPC encoded by pTLPC8 labeledwith fluorescein at twofold molar ratio (left panels; histograms withbroken lines) or FITC-labeled IgG1 (right panels; histograms with brokenlines), both as controls. Both the affinity-improved CD25-specificmutein F92.8 M1.2 E1S and the CD25-specific mAb show significantstaining of the CHO cell line expressing human CD25 while no significantstaining of the CHO cell line expressing human CD154 occurs. Thecontrols wild type TLPC and IgG1 show no significant binding to bothcell lines tested.

FIG. 20 shows a graphical representation of the data from Example 26, inwhich binding measurements with the TLPC muteins S99.3H24, S99.3 C13 andS99.4 F15, respectively, and the prescribed target CD25 as well as theunrelated targets capture mAb, HSA, FCS and captured human IgGFc-fragment were performed by ELISA. Binding of the TLPC muteinsS99.3H24 (closed circle), S99.3 C13 (closed square) and S99.4 F15(closed triangle, respectively) to CD25-Fc (immobilized on the ELISAplate via a capture mAb) was compared with the interaction of therespective muteins with capture mAb (open circle, open square and opentriangle, respectively) as control (also immobilized on the ELISAplate). The TLPC muteins S99.3H24, S99.3 C13 and S99.4 F15 bind CD25 ina concentration-dependent manner, whereas no significant binding signalto the unrelated target capture mAb (open symbols) was detectable.Control binding curves are only shown for this unrelated target, butsimilar results were obtained for the other control targets tested.

FIG. 21 shows a schematic drawing of the expression vector pTLPC14.pTLPC14 codes for a fusion protein of the OmpA signal sequence, a T7detection tag (T7) with a modified tear lipocalin according to (FIG. 7)followed by the C-terminal Strep-tag® II. A relevant segment of thenucleic acid sequence of pTLPC14 is reproduced together with the encodedamino acid sequence in the sequence listing as SEQ ID NO: 25. Thesegment begins with the XbaI restriction site and ends with the HindIIIrestriction site. The vector elements outside this region are identicalwith the vector pASK75, the complete nucleotide sequence of which isexhibited in the German patent publication DE 44 17 598 A1.

FIG. 22 shows a graphical representation of the data from Example 30, inwhich binding measurements of the TLPC monomeric as well as dimericfraction of mutein S100.1 I08 with the prescribed target hCD33-Fc aswell as the unrelated target hCD22 were performed by ELISA. Binding ofthe TLPC mutein S100.1 I08 to hCD33-Fc (closed circles; closedtriangles) was compared with the interaction with hCD22 (open circles;open triangles).The TLPC mutein binds hCD33-Fc in aconcentration-dependent manner, whereas no significant binding signalswere detectable to the unrelated target.

FIG. 23 shows a graphical representation of the data from Example 30, inwhich binding measurements of the TLPC mutein S101.2 A20 with theprescribed target hCD33-Fc as well as the unrelated targets hCD22 andhIgG1 were performed by ELISA. Binding of the TLPC mutein S101.2 A20 tohCD33-Fc (closed circles) was compared with the interaction of themutein with hIgG1 (open circles) and hCD22 (open triangles). The TLPCmutein binds hCD33-Fc in a concentration-dependent manner, whereas nosignificant binding signals were detectable to the unrelated targets.

FIG. 24 shows a graphical representation of the data from Example 30, inwhich binding measurements of the monomeric as well as dimeric fractionof the TLPC mutein S101.2 008 with the prescribed target hCD33-Fc aswell as the unrelated targets hCD22 and hIgG1 were performed by ELISA.Binding of the TLPC mutein S101.2 008 to hCD33-Fc (closed circles;closed squares) was compared with the interaction of the mutein withhIgG1 (open triangles; open diamonds) and hCD22 (open circles; opensquares). The TLPC mutein binds hCD33-Fc in a concentration-dependentmanner, whereas no significant binding signals were detectable to theunrelated targets.

FIG. 25 shows a sensorgram of binding measurements from example 31 inwhich the binding signal measured in RU (=response units) is plottedagainst the time. During injection the TLPC mutein S100.1 I08 associateswith the prescribed target hCD33-Fc. After injection the surface iswashed with running buffer and the mutein dissociates from its target.Association rate and dissociation rate constants (k_(on) and k_(off))were determined using the BIAevaluation software 3.1.

FIG. 26 shows a sensorgram of binding measurements from example 31 inwhich the binding signal measured in RU (=response units) is plottedagainst the time. During injection the TLPC mutein S101.2 A20 associateswith the prescribed target hCD33-Fc. After injection the surface iswashed with running buffer and the mutein dissociates from its target.Association rate and dissociation rate constants (k_(on) and k_(off))were determined using the BIAevaluation software 3.1.

FIG. 27 shows a sensorgram of binding measurements from example 31 inwhich the binding signal measured in RU (=response units) is plottedagainst the time. During injection the TLPC mutein S101.2 O08 associateswith the prescribed target hCD33-Fc. After injection the surface iswashed with running buffer and the mutein dissociates from its target.Association rate and dissociation rate constants (k_(on) and k_(off))were determined using the BIAevaluation software 3.1.

EXAMPLES Example 1 Generation of a Library with 10 Billion IndependentTLPC Muteins

Unless otherwise indicated, established recombinant genetic methods wereused, for example as described in Sambrook et al. (supra).

A random library of TLPC with high complexity was prepared by concertedmutagenesis of in total 17 selected amino acid positions near theN-terminus and in the peptide loops BC, DE, and FG using PCR in multiplesteps according to FIG. 3. The PCR reactions were performed in a volumeof 100 μl in both of the first amplification steps (PCR No. 1, A and B),wherein 10 ng pTLPC6 plasmid-DNA (FIG. 5, SEQ ID NO: 2) was employed astemplate together with 50 pmol of each pair of primers (SEQ ID NO: 3 andSEQ ID NO: 4 or SEQ ID NO: 5 and SEQ ID NO: 6, respectively), which weresynthesized according to the conventional phosphoramidite method. Inaddition, the reaction mixture contained 10 μl 10×Taq buffer (100 mMTris/HCl pH 9.0, 500 mM KCl, 15 mM MgCl₂, 1% v/v Triton X-100) and 2 μldNTP-Mix (10 mM DATP, dCTP, dGTP, dTTP). After bringing to volume withwater, 5 U Taq DNA-polymerase (5 U/μl, Promega) were added and 20 cyclesof 1 minute at 94° C., 1 minute at 62° C. and 1.5 minutes at 72° C. werecarried out in a thermocycler with a heated lid (Eppendorf), followed byan incubation for 5 minutes at 60° C. for final extension. The desiredamplification products were isolated by preparative agarose gelelectrophoresis from GTQ Agarose (Roth) using the Jetsorb DNA extractionkit (Genomed).

For the subsequent amplification step a 2000 μl mixture was prepared,wherein approximately 1000 fmol of both of these respective fragmentswere used as templates, in the presence of 1000 pmol of each of theassembly primers SEQ ID NO: 7, SEQ ID NO: 8 and 20 pmol of the mediatingprimer SEQ ID NO: 9. Both assembly primers had a biotin group at their5′-ends allowing the purification of the PCR-product after BstXIcleavage via streptavidin-coated paramagnetic beads. Additionally, 200μl 10×Taq buffer, 40 μl dNTP-Mix (10 mM DATP, dCTP, dGTP, dTTP), 100 uTaq DNA-polymerase (5 U/μl, Promega) and water were added to bring themixture to the final volume of 2000 μl. The mixture was divided into 100μl aliquots and PCR was performed with 20 cycles of 1 minute at 94° C.,1 minute at 60° C., 1.5 minutes at 72° C., followed by a subsequentincubation for 5 minutes at 60° C. The PCR product was purified usingthe E.Z.N.A. Cycle-Pure Kit (PeqLab).

For cloning purposes, this fragment representing the library of TPLCmuteins in nucleic acid form was first cut with the restriction enzymeBstXI (Promega) according to the instructions of the manufacturer andthen purified by preparative agarose gel electrophoresis as describedabove, resulting in a double stranded DNA-fragment of 303 nucleotides insize. DNA-fragments not or incompletely digested were removed via their5′-biotin tags using streptavidin-coated paramagnetic beads (Merck).

Therefore, 200 μl of the commercially available suspension of theparamagnetic particles in a concentration of 10 mg/ml were washed threetimes with 100 μl TE-buffer. The particles were then drained and mixedwith 100 pmol of the DNA-fragment in 100 μl TE-buffer for 15 minutes atroom temperature. The paramagnetic particles were collected at the wallof the Eppendorf vessel with the aid of a magnet and the supernatantcontaining the purified DNA fragment was recovered for further use inthe following ligation reaction.

The DNA of the vector pTLPC7 (FIG. 4) was cut with BstXI as describedabove and the larger of the two resulting fragments (3907 bp) waspurified by preparative agarose gel electrophoresis. For the ligationreaction, 5.99 μg (30 pmol) of the PCR fragment and 77.3 μg (30 pmol) ofthe vector fragment were incubated in the presence of 833 Weiss Units ofT4 DNA ligase (Promega) in a total volume of 8330 μl (50 mM Tris/HCl pH7.8, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 50 μg/ml BSA) for 24 h at 16° C.The DNA in the ligation mixture was then precipitated by adding 208 μlyeast tRNA (10 mg/ml solution in H₂O (Roche)), 8330 μl 5 M ammoniumacetate, and 33.3 ml ethanol. Incubation at RT for 1 h was followed bycentrifugation (30 minutes, 16000 g, 4° C.). The precipitate washed with5 ml ethanol (70% v/v, RT), centrifuged (10 minutes, 16000 g, 4° C.),and air dried until the DNA pellet appeared glossy and uncolored.Finally, the DNA was dissolved to a final concentration of 200 μg/ml ina total volume of 416.5 μl water.

The preparation of electrocompetent E. coli XL1-Blue (Bullock et al.,supra) was carried out according to the methods described by Tung andChow (Trends Genet. 11 (1995), 128-129) and by Hengen (Trends Biochem.Sci. 21 (1996), 75-76). 11LB-medium was adjusted to an optical densityat 600 nm of OD₆₀₀=0.08 by addition of a stationary XL1-Blue overnightculture and was incubated at 140 rpm and 26° C. in a 21 Erlenmeyerflask. After reaching an OD₆₀₀=0.6, the culture was cooled for 30minutes on ice and subsequently centrifuged for 15 minutes at 4000 g and4° C. The cells were washed twice with 500 ml ice-cold 10% w/v glyceroland finally re-suspended in 2 ml of ice-cold GYT-medium (10% w/vglycerol, 0.125% w/v yeast extract, 0.25% w/v tryptone). The cells werethen aliquoted (200 μl), shock-frozen in liquid nitrogen and stored at−80° C.

The Micro Pulser system (BioRad) was used in conjunction with cuvettesfrom the same vendor (electrode separation 2 mm) for electroporation.All steps were carried out at room temperature employing pre-chilledcuvettes at a temperature of −20° C. Each 10 μl of the DNA solution (2μg) was mixed with 100 μl of the cell suspension, incubated for 1 minuteon ice, and transferred to the pre-chilled cuvette. Electroporation wasperformed (5 ms, 12.5 kV/cm) and the suspension was immediately dilutedin 2 ml SOC-medium, followed by incubation for 60 minutes at 37° C. and140 rpm. Afterwards, the culture was diluted in 412×YT-medium containing100 μg/ml ampicillin (2 YT/Amp) resulting in an OD₅₅₀ of 0.26. Byemploying a total of 78.61 μg ligated DNA about 1.0×10¹⁰ transformantswere obtained in 42 electroporation runs. The transformants were furtherused for preparation of phagemids coding for the library of the TLPCmuteins as fusion proteins as described in Example 7 of the PCTapplication WO 03/029471 or Example 1 of the PCT application WO99/16873.

Example 2 Generation of a Library with 10 Billion Independent TLPCMuteins

A second random library of TLPC with high complexity was prepared byconcerted mutagenesis of selected amino acid positions in the fourpeptide loops AB, CD, EF as well as GH encompassing the naturallipocalin binding pocket at the open end of the lipocalin using PCR inmultiple steps according to FIG. 6. In loop AB a length variation wasintroduced by insertion of either two or four amino acids using the samePCR strategy as described in Example 1, but employing two differentoligodeoxynucleotides (SEQ ID NO: 27 and SEQ ID NO: 28) for preparationof the fragments from PCR A, comprising six or twelve additional randomnucleotides for the insertion of two or four amino acids. In order tostabilize loop AB bearing the four amino acid insertion, the N-terminaland C-terminal anchor positions were fixed by the amino acidsubstitutions V24W, D25S and M31N, N32S encoded by the oligonucleotideSEQ ID NO: 28. The PCR reactions were performed in the same way asdescribed in Example 1 except that in a first amplification step (PCRNo. 1), pTLPC12 plasmid-DNA (FIG. 7, SEQ ID NO: 23) was employed as atemplate together with the primers SEQ ID NO: 26 and SEQ ID NO: 29 toamplify the unextended loop AB, SEQ ID NO: 27 and SEQ ID NO: 29 for theextension by 2 amino acids, or SEQ ID NO: 28 and SEQ ID NO: 29 for theextension by 4 amino acids. This PCR resulted in an amplification of DNAfragments consisting of 336, 342, and 348 base pairs in size whichcomprises nearly the whole structural gene of tear lipocalin with either17 (for loop AB unextended and loop AB extended by 4 amino acids) or 19(for loop AB extended by 2 amino acids) mutated codons. In PCR No. 1Bthe oligonucleotides SEQ ID NO: 30 and SEQ ID NO: 31 were employed inorder to amplify PCR-fragment B. The desired amplification products wereisolated by preparative agarose gel electrophoresis from GTQ Agarose(Roth) using the Wizard SV Gel and PCR Clean-Up System (Promega).

For assembly of the PCR fragments A and B in a subsequent amplificationstep (PCR No. 2), each of the PCR-fragments A was mixed withPCR-fragment B in a separate 1000 μl mixture, wherein approximately 500fmol of both of these respective fragments were used as templates, inthe presence of 500 pmol of each of the assembly primers SEQ ID NO: 33,SEQ ID NO: 34 and 10 pmol of the mediating primer SEQ ID NO: 32. The PCRproducts were purified using the Wizard SV Gel and PCR Clean-Up System(Promega).

For cloning purposes, this fragments representing the library of TPLCmuteins in nucleic acid form were first cut with the restriction enzymeBstXI (Promega) according to the instructions of the manufacturer andthen purified as described above, resulting in double strandedDNA-fragments of 299, 305 and 311 nucleotides in size. DNA-fragments notor incompletely digested were removed via their 5′-biotin tags usingstreptavidin-coated paramagnetic beads (Merck) as described in Example1.

For subsequent ligation of the TLPC muteins from above a 3944 fragmentwas prepared and purified from the DNA of the vector pTLPC12 (FIG. 7) asdescribed in Example 1. For the ligation reaction, 1.97 μg (10 pmol) ofeach PCR fragment and 84 μg (30 pmol) of the vector fragment wereincubated in the presence of 840 Weiss Units of T4 DNA ligase (Promega)in a total volume of 8400 μl (50 mM Tris/HCl pH 7.8, 10 mM MgCl₂, 10 mMDTT, 1 mM ATP, 50 μg/ml BSA) for 38 h at 16° C. The DNA in the ligationmixture was then precipitated by adding 210 μl yeast tRNA (10 mg/mlsolution in H₂O (Roche)), 8400 μl 5 M ammonium acetate, and 33.6 mlethanol. Further processing was performed according to Example 1 andfinally, the DNA was dissolved to a final concentration of 200 μg/ml ina total volume of 420 μl water.

The preparation and transformation of electrocompetent E. coli XL1-Blue(Bullock et al., supra) was carried out according to Example 1. Byemploying a total of 85.97 μg ligated DNA about 0.6×10¹⁰ transformantswere obtained in altogether 42 electroporation runs. The transformantswere further used for preparation of phagemids according to thedescription in Example 7 of the PCT application WO 03/029471 or Example1 of the PCT application WO 99/16873.

Example 3 Phagemid Presentation and Selection of TLPC Muteins AgainstVEGF Employing High Binding Polystyrol Multiwell Plates

For selection of TLPC muteins, 2×10¹² to 1×10¹³ phagemids of the libraryobtained in Example 1 were used. In brief, the phagemids werecentrifuged (21460×g, 4° C., 20 min) and resuspended in 1 ml PBS (4 mMKH₂PO₄, 16 mM Na₂HPO₄, 115 mM NaCl, pH 7.4) containing 50 mMbenzamidine. PBS containing 6% w/v bovine serum albumin (BSA; Roth) and0.3% Tween 20 was used as blocking buffer. Prior to the incubation withthe target protein, phagemids from the library were incubated in bovineserum albumine-blocked polystyrol wells 2 times for 15 minutes for thedepletion of phagemids representing multi-reactive or misfoldedlipocalin mutein. Recombinant human vascular endothelial growth factor(165 aminoacids, rhuVEGF165) produced in insect cells (R&D Systems) wascoated on the polystyrole plates with a concentration of 2.5 μg/ml.After incubation of the blocked phagemids in the coated and blockedwells, adsorbed phagemids were eluted chemically. The adsorbed phagemidswere treated with 300 μl 0.1 M glycine/HCl pH 2.2 per respective wellfor 10 minutes followed by immediate neutralization of the pH of eachelution fraction by mixing it with an appropriate amount of 0.5 M Tris.Beginning with the second enrichment cycle, only half of the combinedphagemid solutions was used for phagemid amplification. After each cycleof selection the titers of the phagemid input, the eighth wash fractionand the eluted phagemids were determined by spot titration. In brief,serial dilutions of the phagemid solution were mixed with E. coliXL1-Blue cells and incubated for 30 min at 37° C. Aliquots of theinfected cells were “spottet” on LB/Amp agar plates and incubated overnight at 37° C. On the next day, the colonies per spot were counted andthe titers of the phagemid solutions (cfu/ml) determined. The phagemidamplification was performed at 22° C.

Four further selection rounds against rhuVEGF165 were carried out inthis way employing the preparation of amplified phagemids from therespective previous enrichment cycle with the exception that only about1×10¹¹ phagemids were utilized beginning with the second enrichmentcycle.

Example 4 Identification of VEGF-Binding TLPC Muteins by Use of aHigh-Throughput ELISA Screening Method

For the analytical production of the TLPC muteins equipped with aC-terminal T7 detection tag (Novagen) followed by a Strep-tag® IIaffinity tag and their characterization by high-throughput ELISAscreening, the vector pTLPC8 (FIG. 8, SEQ ID NO: 24) was constructed.The gene cassette containing the TLPC scaffold between the two BstXIcleavage sites was subcloned from the vector pTLPC7 (FIG. 4, SEQ IDNO: 1) into pTLPC8.

For this purpose the plasmid DNA was isolated from the mixture of the E.coli clones obtained by infection with the phagemids from Example 3eluted as a result of the last selection cycle, using the PlasmidMiniprep Spin kit (Genomed). The DNA was cut with the restriction enzymeBstXI and the smaller of the two fragments was purified by preparativeagarose-gel electrophoresis. The DNA of the vector pTLPC8 was likewisecut with BstXI and the larger of the two fragments (3397 bp) wasisolated in the same way.

For the ligation, each 50 fmol of the two DNA-fragments were mixed with3 Weiss Units T4 DNA ligase (Promega) in a total volume of 20 μl (30 mMTris/HCl pH 7.8, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP), followed byincubation for 2 h at 22° C. E. coli TG1-F (E. coli K12 TG1, which hadlost its episome) was transformed with 5 μl of this ligation mixtureaccording to the CaCl₂-method (Sambrook et al., supra) and plated onLB/Amp agar plates (diameter: 14 cm).

Single E. coli colonies obtained after the transformation harbouring therespective TLPC plasmids coding for the TLPC muteins were picked fromthese agar plates into 70 μl per well 2×YT/Amp in flat bottom 384 wellplates (Greiner) by means of an automated colony picker (Genetix) andgrown overnight at 37° C. at 700 rpm on a benchtop shaker (Bühler) in ahumidified incubator (MMM Medcenter) at 60% relative humidity (rH). Thecultures were diluted 1:100 into 100 μl 2×YT/Amp in round bottom 96 wellplates (Nunc) by means of a 96 pin replicating head (Genetix) and grownfor about 1 h at 37° C. and 60% rH, followed by an incubation for 3 h at22° C. and 60% rH, both at 700 rpm, until the OD₅₅₀ reachedapproximately 0.6. The 384 well plates were kept as “master” plates at−80° C. after adding 25 μl 60% v/v glycerol to each well.

Recombinant TLPC muteins were produced in the 96 well plates by adding20 μl per well of 1.2 μg/ml anhydrotetracyclin in 2×YT (obtained bydiluting a 2 mg/ml stock solution 1:1667 in 2×YT; final concentration0.2 μg/ml) to the bacterial cultures and incubation overnight at 22° C.and 700 rpm at 60% rH. Afterwards, 40 μl of lysis buffer (400 mMNa-borate, pH 8.0, 320 mM NaCl, 4 mM EDTA, 0.3% w/v lysozyme) was addedto each well and the plate was incubated for 1 h at 22° C. and 700 rpmat 60% rH. To minimize non-specific binding interactions in thesubsequent ELISA experiment, obtained crude cell extracts weresupplemented with 40 μl/well PBS containing 10% w/v BSA and 0.05% v/vTween 20 (final concentration 2% w/v BSA) for 1 h at 22° C. and 700 rpmat 60% rH.

For the detection of binding, the crude cell extracts containing theTLPC muteins were tested for their reactivity with the prescribed targetprotein rhuVEGF165 and the unrelated control protein human serumalbumine (HSA, Sigma), respectively, in ELISA experiments. Therefore,wells of black Fluotrac 600 ELISA plates (Greiner; 384 well) were coatedovernight with 20 μl of a solution of rhuVEGF165 at a concentration of2.5 μg/ml in PBS or the control protein at 4 C, 10 μg/ml in PBS. Plateswere washed five times with 100 μl PBS containing 0.05% v/v Tween 20(PBST/0.05) per well with an automated ELISA plate washer (MolecularDevices) leaving a residual volume of 10 μl of the washing buffer ineach well after the last washing step. Residual binding sites wereblocked by incubation with 100 μl PBST/0.05 containing 2% w/v BSA for 2h at room temperature. Afterwards, plates were again washed five timesas described above.

For complex formation between the TLPC muteins and the immobilizedproteins, the wells were incubated with 10 μl of the cell extract fromabove for 1 hour at room temperature. Subsequently, plates were washedagain five times and 10 μl of an anti-T7 monoclonalantibody-HRP-conjugate (Amersham), diluted 1:5000 in PBST/0.05containing 0.5% w/v non-fat dry milk powder (Vitalia), was added to eachwell and incubated for 1 hour at room temperature. Plates were againwashed five times and 10 μl of the fluorogenic HRP-substrate QuantaBlu™(Pierce) was added to detect bound TPLC muteins by means of the attachedanti-T7 monoclonal antibody-HRP-conjugate. After 45 minutes at roomtemperature fluorescence was excited at a wavelength of 320 nm (±12.5nm) and measured at 430 nm (±17.5 nm) in a GENiosPlus plate reader(Tecan).

A selection of 183 TLPC muteins showed significantly higher bindingsignals on the prescribed target protein (rhuVEGF165) compared to theunrelated control protein (HSA) and were subsequently subjected to asecondary high-throughput ELISA screening experiment. Therefore, theseclones were transferred from the 384 well master plates described aboveonto LB/Amp agar, and grown overnight at 37° C. 100 μl 2×YT/Amp in roundbottom 96 well plates (Nunc) was inoculated with single colonies fromthese agar plates and grown overnight at 37° C. at 700 rpm and 60% rH.The cultures were diluted 1:100 into 100 μl 2×YT/Amp in round bottom 96well plates (Nunc) and production of recombinant TLPC muteins as well aspreparation of the bacterial lysates was performed as described above.

For the detection of target-specificity of the TLPC muteins, wells ofblack Fluotrac 600 ELISA plates (Greiner; 384 well) were coatedovernight at 4° C. with 20 μl of a solution of rhuVEGF165 (insect cells,1 μg/ml), or, as controls rhuVEGF165 produced in Escherichia coli(ReliaTech GmbH, 1 μg/ml), recombinant mouse VEGF (rmVEGF164) producedin insect cells (ReliaTech GmbH, 1 μg/ml), HSA, 3% w/v non-fat skimmedmilk powder and StrepTactin (IBA, 10 μg/ml) as well as a conjugate ofRNaseA (Fluka, 10 μg/ml) and digoxigenin in PBS.

This conjugate was prepared by reacting RNaseA at a twofold molar ratioof digoxigenin-3-O-methyl-carbonyl-ε-amidocaproicacid-N-hydroxy-succinimide ester (D)IG-NHS; Roche) according to theinstructions of the manufacturer. Excess reactant was removed from theRNaseA-conjugate by means of size exclusion chromatography using aHiTrap column (Amersham) according to the instructions of themanufacturer employing PBS as running buffer.

After overnight incubation, the plates were washed as described aboveand blocked by the addition of 100 μl/well PBST/0.05 containing 2% w/vBSA at the conditions described above, followed again by washing of theplates. 10 μl of the blocked bacterial lysates of the selected TLPCmuteins mentioned above were transferred to each of the wells coatedwith either rhuVEGF165 or the control proteins and incubated for 1 h atambient temperature. Bound TPLC muteins were detected with anti-17monoclonal antibody-HRP-conjugate and the fluorogenic HRP-substrateQuantaBlu™ as described above.

A selection of 36 TLPC muteins were confirmed on rhuVEGF165 (insectcells) and additionally showed high signals on rhuVEGF165 (E. coli) andrmVEGF164 (insect cells), but did not show binding on unrelated controlproteins (HSA or milk powder).

TLPC muteins with the highest binding signals on the prescribed targetrhuVEGF165 versus the control proteins were selected for sequenceanalysis. Therefore, 4 ml LB/Amp were inoculated with 40 μl of theglycerol stock from the respective well of the 384 well master plate andcultured for subsequent isolation of the plasmid DNA as described at thebeginning of this example. The DNA sequence of the TLPC gene cassettewas elucidated by using the oligodeoxynucleotide SEQ ID NO: 37 as primeron an automated Genetic Analyzer system (Applied Biosystems) accordingto the instructions of the manufacturer employing the Big Dye TerminatorCycle Sequencing Kit (Applied Biosystems).

Six unique sequences of six sequenced clones carried a functionalinsert. The one with the best binding values was named S69.4 O13. Thenucleotide sequence of this clone was translated into the amino acidsequence (SEQ ID NO: 12 in the sequence listing) and those amino acidsdeviating from the modified TLPC encoded by pTLPC8 and the wild-typeTlpc, respectively, are given in Table 1. The clone S69.4O13 was chosenfor the determination of its binding affinity for rhuVEGF165 asdescribed in Example 6. TABLE 1 Sequence characteristics ofanti-rhuVEGF165 TLPC muteins Pos Pos Numbering (Numbering according toaccording to the wild-type experimentally used Tlpc truncated Tlpc TLPC(4001) S69.4 O13 8  4 Glu Gly 9  5 Glu Ile 10  6 Ile Arg 11  7 Gln Arg12  8 Asp Ser 13  9 Val Met 43 39 Thr Leu 45 41 Glu Lys 47 43 Gly His 69 65° Glu Gly 70 66 Lys Arg 72 68 Asp Lys 74 70 Pro Arg 75 71 Gly Lys 9086 Arg Pro 92 88 His Ala 94 90 Lys Arg 97 93 Tyr Val°This amino acid substitution arose from accidental mutation outside therandomized positions.

Example 5 Production of the TLPC Mutein

For the preparative production of the mutein S69.4 O13 described inExample 4, the E. coli K12 strain JM83 harbouring the expression vectorpTLPC8 (FIG. 8, SEQ D NO: 24) encoding this mutein was used for theperiplasmatic production via shake flask expression in an appropriateculture volume of LB-Ampicillin medium according to the protocoldescribed in Schlehuber, S. et al. (J. Mol. Biol. (2000), 297,1105-1120).

When larger amounts of material were needed, the E. coli K12 strainW3110 harbouring the expression vector pTLPC8 encoding this mutein wasused for the periplasmatic production via fermenter cultivation in a0.75 l or 10 l bioreactor based on the protocol described in Schiweck,W., and Skerra, A. (Proteins (1995) 23, 561-565). Fermentation wascarried out at 25° C. The oxygen concentration was maintained at 30%saturation. In a 0.75 l bioreactor, oxygen saturation was kept at 30%via controlling the stirrer speed up to 1500 rpm. In a 10 l reactor,stirrer speed was kept at 480 rpm while supply of air and pure oxygenwas regulated automatically. In fed batch phase 50% w/v Glucose wassupplied stepwise starting with 17.5 ml/h up to 50 ml/h at OD=22.5.

The mutein was purified from the periplasmic fraction in a single stepchromatographic protocol with Strep-Tactin Superflow (IBA) using acolumn of appropriate bed volume and suitable equipment according to themanufacturers' recommendations.

Gel filtration was carried out with Superdex 75 material (AmershamPharmacia Biotech) using a column of appropriate bed volume and suitableequipment according to the manufacturers' recommendations. The monomericfractions were pooled and used for the further characterizations steps.

Example 6 Measurement of the Affinity of the TLPC Muteins for VEGF inELISA

The affinity of the TLPC muteins for VEGF was measured as follows. Inbrief, a dilution series of the mutein S69.4 O13 obtained as describedin Example 5 was tested in an ELISA assay for binding to rhuVEGF165 andthe control protein BSA.

For this purpose, the wells of a black Fluotrac 600 microtiter plate(Greiner; 384 well) were coated with 1 μg/ml rhuVEGF165 (insect cells)and 10 μg/ml BSA (Roth) 0/N at 4° C. and blocked with 2% w/v BSA inPBST/0.1. After a washing step, a subsequent blocking step with 3% w/vmilk powder in PBST and another washing step, a dilution series of themutein S69.4 O13 in PBST covering an appropriate concentration range wasincubated for 1 h at RT in the coated and blocked wells. Bound muteinwas subsequently detected via anti-17 monoclonal antibody-HRP-conjugateand the fluorogenic HRP-substrate QuantaBlu™ as described above. Afteran appropriate incubation time at room temperature, fluorescence wasexcited at a wavelength of 320 nm (±12.5 nm) and measured at 430 nm(±17.5 nm) in a GENiosPlus plate reader.

The curve was fitted by non-linear least squares regression with thehelp of the computer program Kaleidagraph (Synergy software) accordingto the equation [P·L]=([P]_(t)[L]_(t))/(K_(D)+[P]_(t)). Thereby [P]_(t)is the total concentration of immobilized target (in relativefluorescence units), [L]_(t) is the concentration of the applied TLPCmutein, respectively, [P·L] is the concentration of the formed complex(in relative fluorescence units, rFU), and K_(D) is the apparentdissociation constant.

The resulting binding curves versus rhuVEGF165 and BSA are depicted inFIG. 9. The values obtained for the apparent dissociation constants ofthe complex between the TLPC mutein S69.4 O13 and the prescribed targetprotein rhuVEGF165 were identified as 109±34 nM (Table 2). No measurablebinding activity was obtained for the control protein BSA. TABLE 2Affinity binding constants of the TLPC mutein and rhuVEGF165 TLPC muteinK_(D)[nM] rhuVEGF165 K_(D)[nM]BSA VEGF S69.4-O13 109 ± 34 —**No detectable binding activity

Example 7 Phagemid Presentation and Selection of TPLC Muteins Againstthe Extracellular Domain of Human hCD22 Employing Polystyrol MultiwellPlates

For the selection of TLPC muteins the phagemid library from example 1was employed. The selection of TLPC muteins was performed as describedin Example 3. The deviations from the protocol are described in thefollowing: Prior to incubation with the target protein phagemids fromthe library were incubated in BSA-blocked polystyrol wells 2 times for15 minutes each for the depletion of phagemids presenting multi-reactiveor misfolded lipcalin muteins. The extracellular domain of hCD22(Peprotech EC LTD, UK) was coated on polystyrole plates with aconcentration of 5 μg/ml. In the first elution step adsorbed phagemidswere treated with 300 μl 0.1 M glycine/HCl pH 2.2 per respective wellfor 10 minutes followed by immediate neutralization of the pH of eachelution fraction by the addition of an appropriate amount of 0.5 M Tris.The basic elution step was performed with 300 μL 70 mM Triethylamin perrespective well for 10 minutes followed by immediate neutralization ofthe pH of each elution fraction by the addition of an appropriate amountof 1M Tris/HCl, pH 7.4. As a final elution step 300 μl exponentiallygrowing XL1 blue (OD₅₅₀ about 0.5) were transferred in each well andincubated for 30 minutes at 37° C. Beginning with the second enrichmentcycle, only the half of the combined phagemid solutions was used forphagemid amplification as described in Example 3. For the determinationof the phagemid input and the number of eluted phagemids aspot-titration was performed after each cycle of selection from thephagemid used for panning, the 8^(th) wash fraction and the elutedphagemids according to Example 3.

Three further selection rounds against hCD22 were carried out in thisway employing the preparation of amplified phagemids from the respectiveprevious enrichment cycle with the exception that only about 1×10¹¹phagemids were utilized beginning with the second enrichment cycle.

Example 8 Identification of hCD22-Binding TLPC Muteins by Use of aHigh-Throughput ELISA Screening Method

For the analytical production of the hCD22-binding TLPC muteins equippedwith an C-terminal T7 detection tag (Novagen) as well as a Strep-tag® IIaffinity tag and their characterization by high-throughput ELISAscreening, the gene cassette containing the TLPC between the two BstXIcleavage sites was subcloned from the vector pTLPC7 (FIG. 4) into thevector pTLPC8 (FIG. 8). The hCD22-binding TLPC muteins were identifiedby a high-throughput ELISA screening method described in Example 4. TLPCmuteins that bound hCD22 specifically in the primary screening wereselected for more detailed binding analysis in a secondaryhigh-throughput ELISA screening experiment as described in Example 4 aswell.

For the detection of target-specificity of the TLPC muteins, wells ofblack Fluotrac 600 ELISA plates (Greiner; 384 well) were coatedovernight at 4 C with 20 μl of a solution of hCD22 (5 μg/ml, Peprotech)or, as a control, with hCD33-Fc (1 g/ml, R&D Research), hIgG1 (10 μg/ml,Jackson ImmunoResearch), streptactin (10 μg/ml, IBA), human serumalbumin (HSA, 10 μg/ml, Sigma) as well as a conjugate of RNase A (10μg/ml; RNase from Fluka) with digoxin.

All tested TLPC muteins specifically bound hCD22 specific and thenucleotide sequence of their TLPC gene cassette was determined using theoligodeoxynucleotide SEQ ID NO: 37 as a primer on an automated GeneticAnalyzer system (Applied Biosystems) according to the instructions ofthe manufacturer employing the Big Dye Terminator Cycle Sequencing Kit(Applied Biosystems). All sequenced clones exhibited the same sequenceas the clone S76.1H10. The nucleotide sequence of this clone, S76.1H10,was translated into the amino acid sequence and those amino acidsdeviating from the modified TLPC encoded by TLPC 8 (FIG. 8) are given inTable 3. The nucleotide sequence of the clone S76.1H10 is also given asSEQ ID NO: 13. TABLE 3 Sequence characteristics of selected anti-hCD22mutein Pos. Pos Numbering according Numbering according to theexperimentally to wild-type Tlpc used truncated Tlpc TLPC S76.1-H10 8 4Glu Arg 9 5 Glu Trp 10 6 Ile Arg 11 7 Gln Val 12 8 Asp Cys 13 9 Val Trp43 39 Thr Gln 45 41 Glu Asp 47 43 Gly Lys 70 66 Lys Leu 72 68 Asp Asn 7470 Pro Gly 75 71 Gly Val 90 86 Arg Pro 92 88 His Arg 94 90 Lys Ser 97 93Tyr Phe

Example 9 Production of the TLPC Muteins

For the preparative production of the mutein S76.1H10, obtained fromExample 8, the mutagenized coding region between the two BstXI cleavagesites was subcloned from the vector pTLPC7 (FIG. 4) on the expressionplasmid pTLPC8 (FIG. 8). The obtained plasmid thus encoded a fusionprotein of the mutein with the OmpA signal sequence and the T7-tag aswell as the Strep-tag® II at the C-terminus.

Single colonies of E. coli-JM83 and E. coli-W3110, respectively, weretransformed with the plasmid pTLPC8 coding for the TLPC mutein S76.1H10.The shaker flask expression, the 1 liter fermentation, theSA-chromatography and the size exclusion chromatography was performed asdescribed in Example 5. It was found, that the mutein S76.1H10 elutedfrom the size exclusion chromatography (SEC) in two distinct peaks,containing monomeric and dimeric protein, respectively. The bindingaffinity of both protein fractions was determined in an ELISA.

Example 10 Measurement of the Affinity of the TLPC Muteins in ELISA

A dilution series of the mutein S76.1 H10, obtained as described inExample 9, was tested in an ELISA assay for binding to direct coatedhCD22 and the control proteins hCD33-Fc, HAS, and hIgG1.

For this purpose, the wells of black Fluotrac 600 ELISA plates (Greiner;384 well) were coated with 20 μl of hCD22 (5 μg/ml, Peprotech) orcontrol proteins hCD33-Fc (1 μg/ml, R&D Research), HSA (10 μg/ml,Sigma), hIgG1 (10 μg/ml, Jackson ImmunoResearch) O/N at 4° C.

After another washing step, a dilution series of the mutein S76.1H10,obtained in Example 9, in PBST covering an appropriate concentrationrage was added to the coated hCD22 and the control proteins hCD33-Fc,HSA and hIgG1 and incubated for 1 h at RT. Bound mutein was subsequentlydetected via Streptactin-HRP conjugate (IBA) and the fluorogenicHRP-substrate QuantaBlu™ (PIERCE) according to the respectivemanufacturers' recommendations. After an appropriate incubation time atroom temperature, fluorescence was excited at a wavelength of 320 nm(±12.5 nm) and measured at 430 nm (±17.5 nm) in a GENiosPlus platereader.

The curve was fitted by non-linear least squares regression with thehelp of the computer program Kaleidagraph (Synergy software) asdescribed in Example 6.

The resulting binding curves were depicted in FIG. 10 and FIG. 11. Thevalues obtained for the apparent dissociation constants of the complexesbetween the TLPC mutein and the target protein hCD22 as well ascomplexes between the TLPC muteins and the control proteins hCD33-Fc(R&D Systems), human IgG1 (Jackson ImmunoResearch) and human serumalbumin (HSA, Sigma) are summarized in Table 4. No measurable bindingactivity was obtained for the control proteins. TABLE 4 Affinity bindingconstants of the TLPC muteins k_(D) k_(D) k_(D) k_(D) TLPC mutein[nM]hCD22 [nM]hCD33-Fc [nM] hIgG1 [nM] HSA CD22 S76.1  101 ± 3.3 —* —*—* H10 Monomer CD22 S76.1  1.4 ± 0.13 —* —* —* H10 Dimer*No detectable binding activity

Example 11 Phagemid Presentation and Selection of TPLC Muteins Againstthe Extracellular Domain of Human CD25 Employing Polystyrol MultiwellPlates

The target used for the selection of CD25-specific muteins from thephagemid library described in Example 1 and the subsequentcharacterization of these muteins in ELISA experiments was purchasedfrom R&D systems (recombinant human IL-2 R alpha/Fc Chimera).

For the selection of CD25-specific TLPC muteins from the phagemidlibrary described in Example 1, 5 rounds of selection were performed,wherein the capture mAb (Mouse Anti-Human IgG, Fc_(gamma) FragmentSpecific; Jackson ImmunoResearch) was coated on the polystyrole platesat a concentration of 5 μg/ml. After blocking with 2.5% w/v BSA in PBS,CD25-Fc at a concentration of 5 μg/ml was added, incubated for one hourat RT and used for enrichment of CD25-specific phagemids. Adsorbedphagemids were eluted under denaturing conditions with 0.1 M glycine/HClpH 2.2, as described in Example 3.

Example 12 Identification of a CD25-Binding TLPC Mutein by Use of aHigh-Throughput ELISA Screening Method

For the analytical production of the TLPC muteins equipped with aC-terminal T7 detection tag (Novagen) as well as a C-terminal Strep-tag®II affinity tag and their characterization by high-throughput ELISAscreening, the gene cassette between the two BstXI cleavage sites wassub-cloned from the vector pTLPC7 (SEQ ID NO: 1; FIG. 4) into pTLPC8(SEQ ID NO: 24; FIG. 8).

For this purpose the plasmid DNA was isolated from the mixture of the E.coli clones obtained by infection with the phagemids from Example 11eluted as a result of the last selection cycle. Screening forCD25-specific muteins was carried out according to the high-throughputELISA protocol described in Example 4. Crude cell extracts were testedfor binding to the specific target CD25 (immobilized to the microtiterplate as described in Example 11. In parallel, crude cell extracts weretested for binding to the unrelated proteins HSA, Human Gamma Globulin(Jackson ImmunoResearch) and capture mAb, coated at concentrations of 10μg/1 ml, 10 μg/ml, and 5 μg/ml, respectively. Clones with specificbinding properties were confirmed in a secondary high-throughput ELISAassay. In this assay, crude extracts were tested for binding to the sameproteins as used for the primary screening and additional unrelatedproteins (BSA, CD154 (recombinant human sCD40Ligand; Acris; CatalogNumber: PA151XC) and milk, coated at 10 μg/ml, 5 μg/ml and 3%,respectively).

12 clones with a high signal on the specific target and low signals onthe unrelated proteins were selected and the nucleotide sequence of theTLPC gene cassette was determined using the oligodeoxynucleotide SEQ IDNO: 37 as primer on an automated Genetic Analyzer system (AppliedBiosystems) according to the instructions of the manufacturer employingthe Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). Onemutein was found to be enriched during the selection procedure. Thenucleotide sequence of this clone, S67.7 C6, was translated into theamino acid sequence and those amino acids deviating from the modifiedTLPC encoded by pTLPC8 (SEQ ID NO: 24) are given in Table 5. Thenucleotide sequence of S67.7 C6 is also given as SEQ ID NO: 20. TABLE 5Sequence characteristics of a selected TLPC mutein with specificity forCD25 Pos. Pos Numbering according Numbering according to theexperimentally to wild-type Tlpc used truncated Tlpc TLPC8 S67.7 C6 8 4Glu Val 9 5 Glu Gly 10 6 Ile Arg 11 7 Gln Arg 12 8 Asp Gly 13 9 Val Leu43 39 Thr Gly 45 41 Glu Ala 70 66 Lys Gly 72 68 Asp Asn 74 70 Pro Leu 7571 Gly Asp 90 86 Arg His 94 90 Lys Thr 97 93 Tyr Leu

Example 13 Production of the TLPC Mutein

For the preparative production of the mutein S67.7 C6 described inExample 12, the E. coli K12 strain W3110 harbouring the expressionvector pTLPC8 encoding this mutein was used for the periplasmaticproduction via fermenter cultivation as described in Example 5.

The mutein was purified from the periplasmic fraction in a single stepchromatographic protocol with Strep-Tactin Superflow material (IBA)using a column of appropriate bed volume and suitable equipmentaccording to the manufacturers' recommendations.

Gel filtration was carried out with Superdex 75 material (AmershamPharmacia Biotech) using a column of appropriate bed volume and suitableequipment according to the manufacturers' recommendations. The monomericfractions were pooled and used for the further characterization steps.

Example 14 Measurement of the Affinity of the TLPC Mutein for CD25 inELISA

A dilution series of the mutein S67.7 C6 obtained as described inExample 13 was tested in an ELISA assay for binding to captured CD25-Fcand the control proteins capture mAb, HSA, FCS and captured human IgGFc-fragment.

For this purpose, the wells of a black Fluotrac 600 microtiter plate(Greiner; 384 well) were coated with the capture mAb (Mouse Anti-HumanIgG, Fc_(gamma) Fragment Specific; Jackson ImmunoResearch) at aconcentration of 5 μg/ml for 1 h at RT or OIN at 4° C. After a washingstep and a subsequent blocking step with 3% w/v milk powder in PBST,CD25-Fc at a concentration of 5 μg/ml was added and incubated for onehour at RT. In parallel, the unrelated proteins capture mAb, HSA and FCS(Fetal Calf Serum; Invitrogen) were coated at concentrations of 5 μg/ml,10 μl/ml and 10 μg/ml, respectively. In addition, human IgG Fc fragment(Accurate Chemical) was captured at a concentration of 5 μg/ml via thecapture mAb coated at 5 μg/ml.

After another washing step, a dilution series of the mutein S67.7 C6 inPBST covering an appropriate concentration range was added to thecaptured CD25-Fc and the control proteins capture mAb, HSA, FCS andcaptured human IgG Fc fragment and incubated for 1 h at RT. Bound muteinwas subsequently detected via Streptactin-HRP conjugate (MBA) and thefluorogenic HRP-substrate QuantaBlu™ (PIERCE) according to therespective manufacturers' recommendations. After an appropriateincubation time at room temperature, fluorescence was excited at awavelength of 320 nm (±12.5 nm) and measured at 430 nm (±17.5 nm) in aGENiosPlus plate reader.

The curve was fitted by non-linear least squares regression with thehelp of the computer program Kaleidagraph (Synergy software) asdescribed in Example 6.

The resulting binding curves versus captured CD25-Fc and capture mAb aredepicted in FIG. 12. The value obtained for the apparent dissociationconstant of the complex between the TLPC mutein S67.7 C6 and theprescribed target protein CD25-Fc is summarized in Table 6. Nomeasurable binding activity was obtained for the control proteinscapture mAb, HSA, FCS and captured human IgG Fc fragment. TABLE 6Affinity binding constant between the TLPC mutein and CD25-Fc TLPCmutein K_(D) [nM] CD25-Fc S67.7 C6 1178 ± 228

Example 15 Generation of a CHO Cell Line Expressing Human CD25

For the generation of a stable cell line expressing human CD25, CHO-K1cells (DSMZ, No. ACC 110) were transfected with the expression vectorCD25-pcDNA3.1Zeo(+) (SEQ ID NO:10; FIG. 13) encoding human CD25 (NCBIACCESSION NM_(—)000417 [gi:4557666]).

The expression vector CD25-pcDNA3.1Zeo(+) was obtained as described inthe following. The complete coding sequence of human CD25 was amplifiedfrom cDNA of human Peripheral Blood Lymphocytes by PCR using forwardprimer SEQ ID NO:35 and reverse primer SEQ ID NO:36. The PCR productcoding for the full-length protein including the signal peptide wasligated into the cloning vector pCR-BluntII-TOPO (Invitrogen) accordingto the manufacturer's recommendations. The CD25 cDNA was excised fromthe resulting vector by XhoI/HindIII restriction digestion and isolatedby agarose gel electrophoresis as described in Sambrook et al. (supra).The fragment was purified (Wizard SV Clean Up Kit, Promega) and ligatedinto the expression vector pcDNA3.1Zeo(+) (Invitrogen) which had beenlinearized with the same restriction enzymes. E. coli XL1-Blue wastransformed with the resulting expression vector (CD25-pcDNA3.1Zeo(+))and the DNA was extracted and purified using the EndoFree Plasmid MaxiKit (Qiagen).

400,000 CHO-K1 cells (DSMZ, No. ACC 110) grown at 37° C. in DMEMGlutamax I medium (Gibco) containing 10% (v/v) FCS and 5% CO₂ wereplated in 3.5 cm plates and were transfected the following day using 4μg plasmid DNA and 10 μl Lipofectamine2000 (Invitrogen) according to themanufacturer's recommendations. Cells were either transfected withCD25-pcDNA3.1Zeo(+) or pcDNA3.1Zeo(+) as control. One day later, thecells were trypsinized and transferred into five 9.5 cm plates. Thefollowing day, selection started by addition of 200 μg Zeocin per mlmedium. After one week, Zeocin-resistant clones were transferred into 24well plates and subsequently cultured in T25 culture flasks (Greiner).CD25 expression of several clones was analyzed by FACS analysis asdescribed in Example 16. Clones exhibiting the highest expression werekept, stocks were frozen and all further assays were performed withthese cell lines up to passage no. 30.

Example 16 Testing of TLPC Mutein for Specific Binding to a CHO CellLine Expressing Human CD25

The mutein S67.7 C6 was tested for specific binding to a CHO cell lineexpressing human CD25 in a flow cytometry assay. For this purpose, theCD25-pcDNA3.1Zeo(+)- or pcDNA3.1Zeo(+)-transfected CHO cells describedin Example 15 were detached from culture flasks with 0.2% w/v EDTA.Approximately 200,000 cells were resuspended in 30 μl PBS/2% v/v FCS andincubated with 10 μg S67.7 C6 obtained as described in Example 13 andlabeled with fluorescein (Fluorescein-5(6)-carboxamido caproic acidN-succinimidyl ester; Fluka) at an equimolar ratio based on the protocoldescribed in Schlehuber and Skerra (Biol. Chem. (2001) 382, 1335-1342).As a negative control, 10 μg of the recombinant wild type TLPC encodedby pTLPC8 and labeled with fluorescein at an equimolar ratio wasemployed. CD25 expression was confirmed with FITC-labeled anti-CD25 mAb(Acris, DM519F), using FITC-labeled IgG1 (Acris, SM10F) as isotypecontrol. After 30 min incubation on ice, cells were washed twice withPBS/2% v/v FCS prior to analysis by flow cytometry using a FACSCalibur(Becton Dickinson).

Both the CD25-specific mutein S67.7 C6 and the CD25-specific mAb showsignificant staining of the CHO cell line expressing human CD25 while nosignificant staining of the mock-transfected CHO cell line occurs. Thecontrols wild type TLPC and IgG1 show no significant binding to bothcell lines tested. The obtained histograms are depicted in FIG. 14.

Example 17 Generation of an Error-Prone-PCR Library for the AffinityMaturation of a CD25-Specific TLPC Mutein

The CD25-specific mutein S67.7-C06 described in Example 12 was employedfor an affinity maturation procedure. Therefore, a second generationlibrary was prepared, based on mutein S67.7-C06, by employing anerror-prone PCR protocol. This library, already having imprinted thebinding information for CD25, was prepared employing the nucleotideanalogs 8-oxodGTP and dPTP (TEBU-Bio) according to the method describedin literature (Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603). Forthe error-prone amplification reaction the 5′ biotinylatedoligonucleotides SEQ ID NO: 7 and SEQ ID NO: 8 were used together withthe nucleotide analogs. Since these oligodeoxynucleotides are flankingthe BstXI restriction sites, the amplification resulted in pointmutations randomly distributed over the BstXI gene-cassette, whichcomprises most of the structural gene of the TLPC mutein. The PCRproduct was purified using the Wizard SV Gel and PCR Clean-Up System(Promega) and for cloning purposes, this fragment representing theaffinity-matured library of TPLC muteins in nucleic acid form was firstcut with the restriction enzyme BstXI (Promega) according to theinstructions of the manufacturer and then purified as described above,resulting in a double stranded DNA-fragment of 303 nucleotides in size.DNA-fragments not or incompletely digested were removed via their5′-biotin tags using streptavidin-coated paramagnetic beads (Merck) asdescribed in Example 1.

For subsequent ligation of the affinity-matured muteins from above a3907 fragment was prepared and purified from the DNA of the vectorpTLPC7 (FIG. 4) as described in Example 1. For the ligation reaction,3.32 μg (15 pmol) of the PCR fragment and 38.7 μg (15 pmol) of thevector fragment were incubated in the presence of 420 Weiss Units of T4DNA ligase (Promega) in a total volume of 4200 μl (50 mM Tris/HCl pH7.8, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 50 μg/ml BSA) for 48 h at 16° C.The DNA in the ligation mixture was then precipitated by adding 105 μlyeast tRNA (10 mg/ml solution in H₂O (Roche)), 4200 μl 5 M ammoniumacetate, and 16.8 ml ethanol. Further processing was performed accordingto example 1 and at the end the DNA was dissolved to a finalconcentration of 200 g/ml in a total volume of 210 μl of water.

The preparation and transformation of electrocompetent E. coli XL1-Blue(Bullock et al., supra) was carried out according to Example 1. Byemploying a total of 42 μg ligated DNA about 2.6×10⁹ transformants wereobtained in altogether 21 electroporation runs. The transformants werefurther used for preparation of phagemids as described in Example 7 ofthe PCT application WO 03/029471.

Example 18 Phagemid Presentation and Selection of Affinity-ImprovedCD25-Specific TPLC Muteins Employing Polystyrol Multiwell Plates

For the selection of affinity-improved CD25-specific TLPC muteins fromthe error-prone PCR library described in Example 17, 3 rounds ofselection were performed with 2 different strategies (selection strategyA and B, respectively) according to the general method described inExample 3. The deviations from the protocol are described in thefollowing. Prior to the incubation with the target protein, phagemidsfrom the library were incubated in BSA-blocked polystyrol wells 2 timesfor 15 minutes each for the depletion of phagemids presentingmulti-reactive or misfolded lipocalin muteins.

The capture mAb (Mouse Anti-Human IgG, Fc_(gamma) Fragment Specific;Jackson ImmunoResearch) was coated on the polystyrole plates at aconcentration of 5 μg/ml. After blocking with 2.5% w/v BSA in PBS,CD25-Fc at a concentration of 0.063 μg/ml (selection strategy A) or0.016 μg/ml (selection strategy B) was added and incubated for one hourat RT. Adsorbed phagemids were eluted under denaturing conditions and bycompetition using the bacterial strain XL1 blue as described in Example7. In the first, second and third selection cycle about 2×10¹¹, 1×10¹¹and 1×10¹⁰ phagemids were used as input for the enrichment process; and8, 10 and 12 washing cycles were performed, respectively. The phagemidamplification was performed as described in Example 3 except that thephagemids were incubated at 22° C. instead of 26° C.

Example 19 Identification of Affinity-Improved CD25-Specific TLPCMuteins by Use of the Colony Screening Method

For the analytical production of the TLPC muteins as fusion proteinswith the Strep-tag® II and the albumin-binding domain (ABD) and theircharacterization by colony screening, the gene cassette between the twoBstXI cleavage sites was subcloned from the phagemid vector pTLPC7 (SEQID NO: 1; FIG. 4) into pTLPC9 (SEQ ID NO:22; FIG. 15).

For this purpose the plasmid DNA was isolated from the mixture of the E.coli clones obtained by infection with the phagemids of selectionstrategy B from Example 18 eluted as a result of the last selectioncycle. After subcloning of the gene cassette into the screening vectorpTLPC9 and transformation of E. coli K12 TG1-F cells, screening foraffinity-improved, CD25-specific muteins was carried out via thefilter-sandwich colony screening method based on the protocol describedin Schlehuber, S. et al. (supra).

A collection of single clones obtained from O/N cultures in 384 wellmicrotiter plates was spotted in duplicates in an identical pattern onto6 hydrophilic PVDF membranes laid on top of LB/Amp agar plates by meansof a 384 pin head (Genetix). After growth for 4 hours at 37° C.,followed by another incubation step for 2 hours at 22° C., thehydrophilic membranes were placed on top of the hydrophobic membranescoated with HAS, which in turn were placed on top of LB/Amp agar platescontaining 200 μg/l aTc. The culture plates were incubated with thestack of both membranes O/N at 22° C. During this phase the respectiveTLPC muteins were released from the colonies on the upper membranes andwere immobilized via their albumin-binding domain to the HSA on thelower membranes.

For the identification of affinity-improved, CD25-specific muteins, thehydrophobic membranes were screened in parallel with 5 differentconcentrations of CD25-Fc (10 nM, 3 nM, 1 nM, 0.3 nM and 0.1 nM).Mutein/CD25-Fc complexes were detected via anti-human IgG-Fc-HRPconjugate (Peroxidase-conjugated Goat Anti-Human IgG, Fc_(gamma)Fragment Specific; Jackson ImmunoResearch) and the chromogenic DABsubstrate kit for peroxidase (Vector Laboratories) according to therespective manufacturers' recommendations. In parallel, muteinexpression was monitored via Streptactin-HRP conjugate (IBA) and the DABsubstrate kit according to the respective manufacturers'recommendations.

A total of 9 clones from selection strategy B with the highest signal onthe lowest concentration of CD25-Fc was selected and the nucleotidesequence of the respective TLPC gene cassette was determined using theoligodeoxynucleotide SEQ ID NO: 37 as primer on an automated GeneticAnalyzer system (Applied Biosystems) according to the instructions ofthe manufacturer employing the Big Dye Terminator Cycle Sequencing Kit(Applied Biosystems). 8 unique muteins containing a functional insertwere identified. From these, 1 clone was selected for furthercharacterization. The nucleotide sequence of this clone, F92.8 M1.2 E15,was translated into the amino acid sequence and those amino acidsdeviating from modified TLPC encoded by pTLPC9 (SEQ ID NO: 22) are givenin Table 7. The nucleotide sequence of clone F92.8 M1.2 E15 is alsogiven as SEQ ID NO: 21. TABLE 7 Sequence characteristics of a selectedTLPC mutein with improved affinity for CD25 Pos. Pos. Numberingaccording Numbering according to experimentally F92.8 M1.2 to wild-typeTlpc used truncted Tlpc TLPC9 E15 8 4 Glu Val 9 5 Glu Gly 10 6 Ile Arg11 7 Gln Arg 12 8 Asp Gly 13 9 Val Leu 24 19 Thr Ala 43 39 Thr Gly 45 41Glu Ala 50 46 Glu Gly 51 47 Ala Val 70 66 Lys Gly 72 68 Asp Asn 74 70Pro Leu 75 71 Gly Asp 90 86 Arg His 94 90 Lys Thr 97 93 Tyr Leu 99 95Phe Leu

As can be seen from Table 7, the CD25 mutein F92.8 M1.2 E15 carriesamino acid mutations compared to wild type Tlpc at the frameworkpositions 23, 50, and 51.

Example 20 Production of the Affinity-Improved TLPC Mutein Selected bythe Colony Screening Method

For the preparative production of the mutein F92.8 M1.2 E15 described inExample 19, the E. coli K12 strain W3110 harbouring the expressionvector pTLPC9 encoding this mutein was used for the periplasmaticproduction via fermenter cultivation as described in Example 5.

The mutein was purified from the periplasmic fraction in a single stepchromatographic protocol with Strep-Tactin Superflow material (IBA)using a column of appropriate bed volume and suitable equipmentaccording to the manufacturers' recommendations.

Gel filtration was carried out with Superdex 75 material (AmershamPharmacia Biotech) using a column of appropriate bed volume and suitableequipment according to the manufacturers' recommendations. It was found,that the mutein F92.8 M1.2 E15 eluted from the size exclusion column intwo distinct peaks, containing monomeric and dimeric protein,respectively. The monomeric and dimeric fractions were pooled and usedfor the further characterizations steps.

Example 21 Measurement of the Affinity of the Affinity-Improved TLPCMutein for CD25 in ELISA

A dilution series of the mutein F92.8 M1.2 E15 monomeric and dimericfractions obtained as described in Example 20 was tested in an ELISAassay for binding to captured CD25-Fc and the control proteins capturemAb, HSA, FCS and captured human IgG Fc-fragment. The assay wasperformed as described in Example 14, except that 2.5 μg/ml CD25-Fc and2.5 μg/ml human IgG Fc-fragment were used for capturing.

The resulting binding curves versus captured CD25-Fc and capture mAb aredepicted in FIG. 16 and FIG. 17, respectively. The values obtained forthe apparent dissociation constants of the complex between the TLPCmutein F92.8 M1.2 E15 monomer or dimer and the prescribed target proteinCD25-Fc are summarized in Table 8. No measurable binding activity wasobtained for the control proteins capture mAb, HSA, FCS and capturedhuman IgG Fc fragment. TABLE 8 Affinity binding constants between theaffinity-improved TLPC mutein and CD25-Fc TLPC mutein K_(D) [nM] CD25-FcF92.8 M1.2 E15 monomer 131 ± 34  F92.8 M1.2 E15 dimer   6 ± 1.8

Example 22 Generation of a CHO Cell Line Expressing Human CD154

For the generation of a stable cell line expressing human CD154, CHO-K1cells (DSMZ, No. ACC 110) were transfected with the expression vectorCD154-pcDNA3.1Zeo(+) (SEQ ID NO:11; FIG. 18) encoding human CD154 (NCBIACCESSION BC_(—)074950 [gi:49902361]).

The expression vector CD154 pcDNA3.1Zeo(+) was obtained as described inthe following. We obtained the DNA encoding for human CD154 (NCBIACCESSION BC_(—)074950 [gi:49902361]) from a pLXSN vector (BDBiosciences Clontech) in which CD154 was subcloned. The correct sequencefor the complete cDNA was confirmed by sequencing of the plasmidemploying the oligonucleotides SEQ ID NO: 38 and SEQ ID NO: 39. TheDNA-fragment encoding the complete sequence of human CD154 was excisedfrom this vector via restriction digest with XhoI/ApaI and isolated byagarose gel electrophoresis as described in Sambrook et al. (supra). Thefragment was purified (Wizard SV Clean Up Kit, Promega) and ligated intothe expression vector pcDNA3.1Zeo(+) (Invitrogen) which had beenlinearized with the same restriction enzymes. XL1-Blue bacteria weretransformed with the resulting expression vector CD154-pcDNA3.1Zeo(+)and the DNA was extracted and purified using the EndoFree Plasmid MaxiKit (Qiagen).

Growth and transfection of CHO-K1 cells (DSMZ, No. ACC 110) with theexpression vector CD154-pcDNA3.1Zeo(+) was carried out based on theprotocol described in Example 15. CD154 expression of several clones wasanalyzed by FACS analysis as described in Example 23. Clones exhibitingthe highest expression were used for all further assays up to passageno. 30.

Example 23 Testing of Affinity-Improved TLPC Mutein for Specific Bindingto CHO Cell Line Expressing Human CD25

The mutein F92.8 M1.2 E15 was tested for specific binding to a CHO cellline expressing human CD25 in a flow cytometry assay. For this purpose,the CD25-pcDNA3.1 Zeo(+)- or CD154-pcDNA3.1Zeo(+)-transfected CHO cellsdescribed in Examples 15 and 22, respectively were detached from cultureflasks with 0.2% w/v EDTA. Approximately 200,000 cells were resuspendedin 30 μl PBS/2% v/v FCS and incubated with 2.5 μg of monomeric F92.8M1.2 E15 fraction obtained as described in Example 20 and labeled at atwofold molar ratio with fluorescein (Fluorescein-5(6)-carboxamidocaproic acid N-succinimidyl ester; Fluka) based on the protocoldescribed in Schlehuber and Skerra (supra). As a negative control, 2.5μg of the recombinant wild type TLPC encoded by pTLPC8 and labeled withfluorescein at a twofold molar ratio was employed. CD25 expression wasconfirmed with FITC-labeled anti-CD25 mAb (Acris, DM519F), usingFITC-labeled IgG1 (Acris, SM10F) as isotype control. After 30 minincubation on ice, cells were washed twice with PBS/2% v/v FCS prior toanalysis by flow cytometry using a FACSCalibur (Becton Dickinson).

Both the affinity-improved CD25-specific mutein F92.8 M1.2 E15 and theCD25-specific mAb show significant staining of the CHO cell lineexpressing human CD25 while no significant staining of the CHO cell lineexpressing human CD154 occurs. The controls wild type TLPC and IgG1 showno significant binding to both cell lines tested. The obtainedhistograms are depicted in FIG. 19.

Example 24 Identification of Affinity-Improved CD25-Binding TLPC Muteinsby Use of a High-Throughput ELISA Screening Method

For the analytical production of the affinity-improved TLPC muteinsequipped with a C-terminal T7 detection tag (Novagen) as well as aC-terminal Strep-tag® II affinity tag and their characterization byhigh-throughput ELISA screening, the gene cassette between the two BstXIcleavage sites was subcloned from the vector pTLPC7 (FIG. 4) into pTLPC8(FIG. 8).

For this purpose the plasmid DNA was isolated from the mixture of the E.coli clones obtained by infection with the phagemids from Example 18eluted as a result of the last selection cycle. Screening foraffinity-improved, CD25-specific muteins was carried out according tothe high-throughput ELISA protocol described in Example 4. Crude cellextracts were tested for binding to CD25-Fc captured at differentconcentrations (5 μg/ml, 1 μg/ml, 0.2 μg/ml, 0.04 μg/ml and 0.008 μg/ml,respectively). In parallel, crude cell extracts were tested for bindingto human IgG Fc-fragment (Accurate Chemical) captured at a concentrationof 5 μg/ml via the capture mAb which was coated at 5 μg/ml. Clonesshowing specific binding properties and retaining high signals on thelowest target concentrations were confirmed in a secondaryhigh-throughput ELISA. In this assay, crude extracts were tested forbinding to CD25-Fc captured at 1 μg/ml and 0.1 μg/ml. In addition, crudeextracts were tested for binding to the unrelated proteins capture mAb,HSA, CD154 and Human Gamma Globulin (Jackson ImmunoResearch), coated at5 μg/1 ml, 10 μg/ml, 5 μg/ml and 10 μg/ml, respectively.

A total of 13 clones from both selection strategies giving rise to ahigh signal at the lowest concentrations of captured CD25-Fc and lowsignals on the unrelated proteins was selected and the nucleotidesequence of the respective TLPC gene cassette was determined using theoligodeoxynucleotide SEQ ID NO: 37 as primer on an automated GeneticAnalyzer system (Applied Biosystems) according to the instructions ofthe manufacturer employing the Big Dye Terminator Cycle Sequencing Kit(Applied Biosystems). 7 unique muteins containing a functional insertwere identified. From these, 3 clones were selected for furthercharacterization. The nucleotide sequence of these clones, S99.3H24 andS99.3 C13 derived from selection strategy A and S99.4 F15 obtained fromselection strategy B, was translated into the amino acid sequence andthose amino acids deviating from modified TLPC encoded by pTLPC8 (SEQ IDNO: 24) are given in Table 9. The nucleotide sequence of clonesS99.3H24, S99.3 C13 and S99.4 F15 is also given as SEQ ID NO: 17, SEQ IDNO: 18 and SEQ ID NO: 19, respectively. TABLE 9 Sequence characteristicsof selected TLPC muteins with improved affinity for CD25 Pos. Pos.Numbering Numbering according to according to wild-type exp used Tlpctruncted Tlpc TLPC8 S99.3 H24 S99.3 C13 S99.4 F15 8 4 Glu Val Val Val 95 Glu Gly Gly Gly 10 6 Ile Lys Arg Arg 11 7 Gln Arg Arg Arg 12 8 Asp GlyGly Gly 13 9 Val Leu Leu Leu 28 24 Phe Phe Ser Ser 32 28 Asn Asp Asn Asn43 39 Thr Gly Gly Gly 45 41 Glu Ala Ala Ala 67 63 Val Val Ala Val 70 66Lys Gly Gly Gly 72 68 Asp Asn Asn Asn 74 70 Pro Leu Leu Leu 75 71 GlyAsp Asp Asp 86 82 Ala Ala Val Ala 90 86 Arg His His His 91 87 Ser ProPro Pro 94 90 Lys Thr Thr Thr 97 93 Tyr Leu Leu Leu

As can be seen from Table 9, the Tlpc muteins identified from theaffinity maturation contained not only mutations in the binding site atthe closed end of the β-barrel structure but also mutations in thepeptide segments forming the natural lipocalin binding pocket (hereresidues 28, 32 of AB peptide loop) and at positions of the frameworkregion (positions 67 and 86 of the Tlpc sequence, respectively).

Example 25 Production of the Affinity-Improved TLPC Mutein Selected bythe High-Throughput ELISA Screening Method

For the preparative production of the muteins S99.3H24, S99.3 C13 andS99.4 F15 described in Example 24, the E. Coli K12 strain W3110harbouring the expression vector pTLPC8 encoding these muteins was usedfor the periplasmatic production via fermenter cultivation as describedin Example 5.

The mutein was purified from the periplasmic fraction in a single stepchromatographic protocol with Strep-Tactin Superflow material (IBA)using a column of appropriate bed volume and suitable equipmentaccording to the manufacturers' recommendations.

Gel filtration was carried out with Superdex 75 material (AmershamPharmacia Biotech) using a column of appropriate bed volume and suitableequipment according to the manufacturers' recommendations. The monomericfractions were pooled and used for the further characterizations steps.

Example 26 Measurement of the affinity of the affinity-improved TLPCmutein for CD25 in ELISA

A dilution series of the muteins S99.3H24, S99.3 C13 and S99.4 F15obtained as described in Example 25 was tested in an ELISA assay forbinding to captured CD25-Fc and the control proteins capture mAb, HSA,FCS and captured human IgG Fc-fragment.

The assay was performed as described in Example 14, except that 2.5μg/ml CD25-Fc and 2.5 μg/ml human IgG Fc fragment were used forcapturing.

The resulting binding curves versus captured CD25-Fc and capture mAb aredepicted in FIG. 20. The values obtained for the apparent dissociationconstants of the complex between the TLPC muteins S99.3H24, S99.3 C13and S99.4 P15 and the prescribed target protein CD25-Fc are summarizedin Table 10. No measurable binding activity was obtained for the controlproteins capture mAb, HSA, FCS and captured human IgG Fc-fragment. TABLE10 Affinity binding constants between the affinity-improved TLPC muteinsand CD25-Fc TLPC mutein K_(D) [nM] CD25-Fc S99.3 H24 302 ± 46  S99.3 C13307 ± 43  S99.4 F15  22 ± 2.4

Example 27 Phagemid Presentation and Selection of TPLC Muteins Againstthe Extracellular Domain of Human CD33-Fc Employing Polystyrol MultiwellPlates and Protein A Magnetic Beads

For the selection of TLPC muteins the phagemid library, as described inExample 2, was used. The selection of TLPC muteins employing polystyrolmultiwell plates was performed as described in Example 3. The deviationsfrom the protocol are described in Example 7. The target hCD33-Fc (R&DResearch) was directly coated on the polystyrol plates with aconcentration of 1 μg/ml.

The selection of TLPC muteins was performed employing protein A beads(Dynabeads Protein A, Dynal) essentially following the instructions ofthe manufacturer. BSA was chosen as blocking agent for phagemids andtarget. The phagemids were eluted under acidic (0.1 M glycin/Hcl pH 2.2;10 min RT; neutralization with 0.5 M Tris-base) and/or basic conditions(70 mM triethylamine; 10 min RT; neutralization with 1 M Tris/HCl, ph7.4) followed by a final bacterial elution step, as described in Example7.

The deviations from the protocol are described in Example 7 with theexception, that prior to incubation with the target protein phagemidsfrom the library were incubated with 100 μl of BSA-blocked protein Abeads, 2 times for 15 minutes each, for the depletion of phagemidspresenting multi-reactive or misfolded lipocalin muteins.

Four rounds of selection against hCD33-Fc were carried out in this wayemploying the preparation of amplified phagemids from the respectiveprevious enrichment cycle with the exception that only about 1·10¹¹phagemids were utilized beginning with the second cycle.

Example 28 Identification of hCD33-Binding TLPC Muteins by Use of aHigh-Throughout ELISA Screening Method

For the analytical production of the hCD33-binding TLPC muteins equippedwith an N-terminal T7 detection tag (Novagen) as well as a Strep-tag® IIaffinity tag at the C-terminus and their characterization byhigh-throughput ELISA screening, the gene cassette containing the TLPCbetween the two BstXI cleavage sites was subcloned from the vectorpTLPC12 (FIG. 1) into the vector pTLPC14 (FIG. 21). The hCD33-bindingTLPC muteins were identified by a high-throughput ELISA screening methodas described in example 4. TLPC muteins that bound hCD33 specifically inthe primary screening were selected for more detailed binding analysisin a secondary high-throughput ELISA screening experiment as describedin the same example.

For the detection of target-specificity of the recombinant TLPC muteins,wells of black Fluotrac 600 ELISA plates (Greiner; 384 well) were coatedovernight at 4° C. with 20 μl of a solution of AffiniPure mouseanti-human IgG Fc_(Gamma) fragment-specific antibody (5 μg/ml, JacksonImmunoResearch) and as a control, with hCD22 (5 μg/ml, Peprotech), hIgG1(10g/ml, Jackson ImmunoResearch), streptactin (10 μg/ml, BA), humanserum albumin (10 μg/ml, Sigma) as well as a conjugate of RNase A (10μg/ml; RNase from Fluka) and digoxin. The target hCD33-Fc was capturedvia the anti-human IgG Fc_(Gamma) fragment-specific antibody for 1 h atRT.

Multiple TLPC muteins turned out to bind hCD33-Fc specific and thenucleotide sequence of the TLPC gene cassette was determined fromseveral clones using the oligodeoxynucleotide SEQ ID NO: 37 as a primeron an automated Genetic Analyzer system (Applied Biosystems) accordingto the instructions of the manufacturer employing the Big Dye TerminatorCycle Sequencing Kit (Applied Biosystems).

The sequencing of clones revealed from the polystyrol multiwell panningrevealed 4 different lipocalin muteins. Two of them were analysedfurther. The nucleotide sequence of these clones was translated into theamino acid sequence and those amino acids deviating from the modifiedTLPC encoded by TLPC14 (FIG. 21) are given in Table 11. The nucleotidesequences of these lipocalin muteins, named S101.2 O08 and S101.2 A20,are given as SEQ ID NO 16, and SEQ ID NO 15, respectively.

The sequencing of clones selected from the protein A bead panningrevealed two different lipocalin muteins. The nucleotide sequence of theclone S100.1-I08, chosen for further analysis, was translated into theamino acid sequence and those amino acids deviating from the modifiedTLPC encoded by TLPC14 (FIG. 21) are given in Table 11. The nucleotidesequence is also given as SEQ ID NO: 25. TABLE 11 Sequencecharacteristics of selected anti-hCD33-Fc muteins Pos. Numberingaccording to the wild type Tlpc TLPC S101.2-A20 S101.2-O08 S100.1-I08 25Asp Val Pro Gly +4 — — — +3 — — — +2 — Asp — +1 — Leu — 26 Arg His SerSer 27 Glu Gly Leu Gly 28 Phe Val Thr Ser 29 Pro His Leu Ile 30 Glu AspGln Cys 31 Met Leu Ala Thr 32 Asn Phe Thr Cys 33 Leu Leu Ala Ser 56 LeuPhe Phe Val 57 Ile Gly Gly Val 58 Ser Asn Tyr Arg  65° Lys Asn Lys Lys83 Lys Asn Asn Asn 105  Leu His Leu Val 106  His Met Met Met 108  LysTrp Val Leu 109  Pro Thr Leu Pro°These amino acid substitutions arose from accidental mutations outsidethe randomized positions.+2, +4 describe the insertion of two or 4 amino acids in loop 1 of theTLPC library described in Example 2.

Example 29 Production of the TLPC Muteins

For the preparative production of the anti hCD33 muteins S100.1 I08,S101.2 A20 and S101.2 O08 obtained from Example 28 the mutagenizedcoding region between the two BstXI cleavage sites was subcloned fromthe vector pTLPC12 (FIG. 7) on the expression plasmid pTLPC14 (FIG. 21).The obtained plasmid thus encoded a fusion protein of the mutein withthe OmpA signal sequence and the T7-tag at the N-terminus as well as theStrep-tag® II at the C-terminus.

Single colonies of E. coli-W3110 (fermentation) or E. coli-JM83 (shakerflask expression) were transformed with the pTLPC14 plasmids coding forthe TLPC muteins S100.1 I08, S101.2 A20 or S101.2 O08, respectively. Theshaker flask expression, the 1 l fermentation, the SA-chromatography andthe size exclusion chromatography (SEC) were performed as described inExample 5. The SEC revealed a dimeric and a monomeric protein fractionfor the clones S100.1-I08 and S101.2 O08. The binding affinity ofmonomeric and dimeric fraction was separately determined in an ELISA.

Example 30 Measurement of the Affinity of the TLPC Muteins in ELISA

For the determination of binding affinity of the selected TLPC muteinsfrom Example 28 for the prescribed protein target hCD33-Fc as well asthe unrelated control proteins in an ELISA the wells of black Fluotrac600 ELISA plates (Greiner; 384 well) were coated with 20 μl hCD33-Fc (1μg/ml), AffiniPure mouse anti-human IgG Fc Gamma fragment-specificantibody (5 μg/ml, Jackson ImmunoResearch) and as a control, with hIgG1(10 μg/ml, Jackson ImmunoResearch) O/N at 4° C. The targets hCD33-Fc (1μg/ml, R&D Research) and hCD22-Fc (1 μg/ml, Peprtoech) were captured viathe AffiniPure mouse anti-human IgG Fc_(Gamma) fragment-specificantibody for 1 h at RT. Afterwards, the ELISA was performed with theTLPC muteins from Example 29 as described in Example 10.

The resulting binding curves were fitted as described in Example 10 andare depicted in FIGS. 22-24. The values obtained for the apparentdissociation constants of the complexes between the TLPC muteins and thetarget protein hCD33-Fc as well as complexes between the TLPC muteinsand the control proteins hCD22-Fc (R&D Systems) and HSA (Sigma) aresummarized in Table 12. TABLE 12 Affinity binding constants of the TLPCmuteins K_(D)[nM]hCD33- K_(D)[nM]hCD22- K_(D)[nM] TLPC mutein, monomerFc Fc hIgG1 CD33 S101.2 A20  5.2 ± 0.72 —* —* CD33 S101.2 O08 187 ± 26.9—* —* (monomer) CD33 S100.1 I08 131 ± 38.8 —* ND (monomer) CD33 S101.2O08  83 ± 13.7 —* —* (dimer) CD33 S100.1 I08 (dimer) 6.3 ± 1.4  —* ND*No detectable binding activity;ND = not determined

Example 31 Measurement of the Affinity of the TLPC Muteins in BIAcore

14000 response units (RU) AffiniPure mouse anti-human IgG Fc Gammafragment-specific antibody (Jackson Immunoresearch) were coupled byamine coupling to a CM5 sensor chip (Biacore) according to themanufacturers' recommendations. 3000RU hCD33-Fc (R&D research) werecaptured to this surface by injecting 10 μl of a 0.2 mg/ml hCD33-Fcsolution at a flow rate of 2 μl/min. BBS (10 mM HEPES, 150 mM NaCl, 2 mMEDTA, 0.005% v/v Tween pH 7.4) was used as running buffer. All sampleswere diluted in this running buffer. The TLPC muteins, obtained inexample 29, were added to the hCD33-Fc captured surface by injection ofa 40 μl sample with a 20 μl/min flow rate. The solutions of added TLPCmutein were 10 μM and 6.4 μM for S101.2 A20 and S101.2 O08,respectively. The surface of the chip was regenerated with 10 mM HClfollowed by recoupling of hCD33-Fc before the next lipocalin mutein wasmeasured. All measurements were performed on a BIAcore X apparatus. Todetermine the binding affinity of S100.1 I08 2000 RU of hCD33-Fc werecaptured to the surface described above and the solution of thelipocalin mutein added had a concentration of 5 μM. The obtained bindingcurves were fitted using the BIAevaluation software 3.1 from Biacore andare shown in FIGS. 25-27. The resulting affinity binding constants ofthe TLPC muteins are summarized in Table 13. TABLE 13 Affinity bindingconstants of the TLPC muteins TLPC mutein k_(on) [M⁻¹s⁻¹] k_(off) [s⁻¹]K_(D) [nM]hCD33-Fc CD33 S101.2 A20 2.3 × 10⁴ 2.0 × 10⁻³ 87 CD33 S101.2O08 1.3 × 10⁴ 1.9 × 10⁻³ 146 CD33 S100.1 I08 1.5 × 10⁴ 8.5 × 10⁻⁴ 57

1-30. (canceled)
 30. A mutein of human tear lipocalin, wherein themutein comprises at least two mutated amino acid residues with respectto the wild type amino acid sequence of mature human tear lipocalin atany sequence position in each of the four peptide segments that areformed by the sequence positions 7-14, 43-49, 70-77, and 87-97 of thelinear polypeptide sequence of human tear lipocalin, and wherein themutein binds a given non-natural target of human tear lipocalin withdetectable affinity.
 31. The mutein of claim 30, wherein the muteincomprises amino acid mutations at least any 12-16 of the sequencepositions 8, 9, 10, 11, 12, 13, 43, 45, 47, 70, 72, 74, 75, 90, 92, 94,and 97 of the linear polypeptide sequence of human tear lipocalin.
 32. Amutein of human tear lipocalin, wherein the mutein comprises at leasttwo mutated amino acid residues with respect to the wild type amino acidsequence of mature human tear lipocalin at any sequence position in eachof the four peptide segments that are formed by the sequence positions24-36, 53-66, 79-84, and 103-110 of the linear polypeptide sequence ofhuman tear lipocalin, and wherein the mutein binds a given non-naturaltarget of human tear lipocalin with detectable affinity.
 33. The muteinof claim 32, wherein the mutein comprises amino acid mutations at leastany 16 of the sequence positions 25, 26, 27, 28, 29, 30, 31, 32, 33, 56,57, 58, 83, 105, 106, 108 and 109 of the linear polypeptide sequence ofhuman tear lipocalin.
 34. The mutein of claim 32, further comprisingamino acid mutations at least any 12-16 of the sequence positions 7-14,43-49, 70-77, and 87-97 of the linear polypeptide sequence of human tearlipocalin.
 35. The mutein of claim 32, comprising amino acid mutationsat least 12-16 of any of the sequence positions 8, 9, 10, 11, 12, 13,43, 45, 47, 70, 72, 74, 75, 90, 92, 94, and 97 of the linear polypeptidesequence of human tear lipocalin.
 36. A mutein of human tear lipocalin,wherein the mutein comprises at least two mutated amino acid residueswith respect to the wild type amino acid sequence of mature human tearlipocalin at any sequence position in each of the four peptide segmentsthat are formed by the sequence positions 7-14, 43-49, 70-77, and 87-97of the linear polypeptide sequence of human tear lipocalin, wherein themutein comprises at least two mutated amino acid residues with respectto the wild type amino acid sequence of mature human tear lipocalin atany sequence position in each of the four peptide segments that areformed by sequence positions 24-36, 53-66, 79-84, and 103-110 of thelinear polypeptide sequence of human tear lipocalin, and wherein themutein binds at least one given non-natural target of human tearlipocalin with detectable affinity.
 37. The mutein of claim 30, whereinthe mutein is conjugated to a label selected from the group consistingof organic molecules, enzyme labels, radioactive labels, colored labels,fluorescent labels, chromogenic labels, luminescent labels, haptens,digoxigenin, biotin, metal complexes, metals, and colloidal gold. 38.The mutein of claim 30, wherein the mutein is fused at its N-terminus orits C-terminus to a protein, a protein domain or a peptide.
 39. Anucleic acid molecule comprising a nucleotide sequence encoding a muteinof claim
 30. 40. The nucleic acid molecule of claim 39 comprised in avector.
 41. The nucleic acid molecule of claim 40 comprised in aphagemid vector.
 42. A host cell containing a nucleic acid molecule ofclaim
 39. 43. A method for the generation of a mutein of human tearlipocalin of claim 30, comprising: (a) subjecting a nucleic acidmolecule encoding human tear lipocalin to mutagenesis at two or moredifferent codons of any sequence position in each of the four peptidesegments that are formed of the sequence positions 7-14, 43-49, 70-77,and 87-97 of the linear polypeptide sequence of human tear lipocalin,(b) expressing at least one mutein nucleic acid molecules obtained in(a) in a suitable expression system, and (c) enriching at least onemutein having a detectable binding affinity for a given target by meansof selection and/or isolation.
 44. A method for the generation of amutein of human tear lipocalin of claim 32, comprising: (a) subjecting anucleic acid molecule encoding human tear lipocalin to mutagenesis attwo or more different codons of any sequence position in each of thefour peptide segments that are formed of the sequence positions 24-36,53-66, 79-84, and 103-110 of the linear polypeptide sequence of humantear lipocalin, (b) expressing at least one mutein nucleic acidmolecules obtained in (a) in a suitable expression system, and (c)enriching at least one mutein having a detectable binding affinity for agiven target by means of selection and/or isolation.
 45. A method forthe production of a mutein according to claim 30, wherein the mutein, afragment of the mutein or a fusion protein of the mutein and anotherpolypeptide is produced starting from the nucleic acid coding for themutein by means of genetic engineering methods.
 46. The method of claim45, wherein the mutein is produced in a bacterial or eukaryotic hostorganism and is isolated from this host organism or its culture.
 47. Amethod for the production of a mutein according to claim 30, wherein themutein, a fragment of the mutein or a fusion protein of the mutein andanother polypeptide is produced by peptide synthesis.
 48. Apharmaceutical composition comprising at least one mutein of claim 30.49. A method of detection a given target in vitro, comprising the stepsof: (a) contacting a mutein as defined in claim 30 with a test samplesupposed to contain said target, and (b) detecting of the mutein/targetcomplex by a suitable signal.
 50. The method of claim 49, wherein thegiven target is a protein or protein domain, a peptide, a nucleic acidmolecule, an organic molecule or a metal complex.
 51. The method ofclaim 49, wherein the detection is carried out for validation of theprotein as pharmacological drug target.
 52. A method of separating agiven target from a sample in vitro, comprising: (a) contacting a muteinas defined in claim 30 with the sample supposed to contain said target,and (b) separating the mutein/target complex from the sample.
 53. Themethod of claim 49, wherein the mutein/target complex is bound onto asolid phase.
 54. A method of targeting a compound to a preselected sitein vitro comprising (a) contacting a mutein as defined in claim 30 withsaid compound, and (b) delivering the mutein/compound complex to thepreselected site.
 55. A pharmaceutical composition comprising at leastone mutein of claim 32.